Guanidinium functionalized intermediates

ABSTRACT

The present invention provides oligomers which are specifically hybridizable with a selected sequence of RNA or DNA wherein at least one of the nucleoside moieties of the oligomer is modified to include a guanidinium group. These oligomers are useful for diagnostic, therapeutic and investigative purposes.

RELATED APPLICATION DATA

This patent application is a divisional application of application Ser.No. 09/612,531, filed Jul. 7, 2000, now U.S. Pat. No. 6,539,639, whichis a continuation-in-part of application Ser. No. 09/349,040, filed Jul.7, 1999, now U.S. Pat. No. 6,593,466, the contents of which areincorporated herein by reference.

FIELD OF THE INVENTION

The present invention relates to monomers and oligomers containingguanidinium moieties and methods of preparing such oligomers. Theoligomers of the present invention are used for investigative andtherapeutic purposes.

BACKGROUND OF THE INVENTION

It is well known that most of the bodily states in mammals, includingmost disease states, are affected by proteins. Classical therapeuticmodes have generally focused on interactions with such proteins in aneffort to moderate their disease-causing or disease-potentiatingfunctions. However, recently, attempts have been made to moderate theactual production of such proteins by interactions with molecules thatdirect their synthesis, such as intracellular RNA. By interfering withthe production of proteins, maximum therapeutic effect and minimal sideeffects may be realized. It is the object of such therapeutic approachesto interfere with otherwise modulate gene expression leading toundesired protein formation.

One method for inhibiting specific gene expression is the use ofoligonucleotides. Oligonucleotides are now accepted as therapeuticagents with great promise. Oligonucleotides are known to hybridize tosingle-stranded DNA or RNA molecules. Hybridization is thesequence-specific base pair hydrogen bonding of nucleobases of theoligonucleotide to the nucleobases of the target DNA or RNA molecule.Such nucleobase pairs are said to be complementary to one another. Theconcept of inhibiting gene expression through the use ofsequence-specific binding of oligonucleotides to target RNA sequences,also known as antisense inhibition, has been demonstrated in a varietyof systems, including living cells (f or example see: Wagner et al.,Science (1993) 260: 1510-1513; Milligan et al., J. Med. Chem., (1993)36:1923-37; Uhlmann et al., Chem. Reviews, (1990) 90:543-584; Stein etal., Cancer Res., (1988) 48:2659-2668).

The events that provide the disruption of the nucleic acid function byantisense oligonucleotides (Cohen in Oligonucleotides: AntisenseInhibitors of Gene Expression, (1989) CRC Press, Inc., Boca Raton, Fla.)are thought to be of two types. The first, hybridization arrest, denotesthe terminating event in which the oligonucleotide inhibitor binds tothe target nucleic acid and thus prevents, by simple steric hindrance,the binding of essential proteins, most often ribosomes, to the nucleicacid. Methyl phosphonate oligonucleotides: Miller and Ts'O, Anti-CancerDrug Design, 1987, 2:117-128, and α-anomer oligonucleotides are the twomost extensively studied antisense agents which are thought to disruptnucleic acid function by hybridization arrest.

The second type of terminating event for antisense oligonucleotidesinvolves the enzymatic cleavage of the targeted RNA by intracellularRNase H. A 2′-deoxyribofuranosyl oligonucleotide or oligonucleotideanalog hybridizes with the targeted RNA and this duplex activates theRNase H enzyme to cleave the RNA strand, thus destroying the normalfunction of the RNA. Phosphorothioate oligonucleotides are the mostprominent example of an antisense agent that operates by this type ofantisense terminating event.

Oligonucleotides may also bind to duplex nucleic acids to form triplexcomplexes in a sequence specific manner via Hoogsteen base pairing (Bealet al., Science, (1991) 251:1360-1363; Young et al., Proc. Natl. Acad.Sci. (1991) 88:10023-10026). Both antisense and triple helix therapeuticstrategies are directed towards nucleic acid sequences that are involvedin or responsible for establishing or maintaining disease conditions.Such target nucleic acid sequences may be found in the genomes ofpathogenic organisms including bacteria, yeasts, fungi, protozoa,parasites, viruses, or may be endogenous in nature. By hybridizing toand modifying the expression of a gene important for the establishment,maintenance or elimination of a disease condition, the correspondingcondition may be cured, prevented or ameliorated.

In determining the extent of hybridization of an oligonucleotide to acomplementary nucleic acid, the relative ability of an oligonucleotideto bind to the complementary nucleic acid may be compared by determiningthe melting temperature of a particular hybridization complex. Themelting temperature (T_(m)) a characteristic physical property of doublehelices, denotes the temperature (in degrees centigrade) at which 50%helical (hybridized) versus coil (unhybridized) forms are present. T_(m)is measured by using the UV spectrum to determine the formation andbreakdown (melting) of the hybridization complex. Base stacking, whichoccurs during hybridization, is accompanied by a reduction in UVabsorption (hypochromicity). Consequently, a reduction in UV absorptionindicates a higher T_(m). The higher the T_(m), the greater the strengthof the bonds between the strands.

Oligonucleotides may also be of therapeutic value when they bind tonon-nucleic acid biomolecules such as intracellular or extracellularpolypeptides, proteins, or enzymes. Such oligonucleotides are oftenreferred to as “aptamers” and they typically bind to and interfere withthe function of protein targets (Griffin et al., Blood, (1993),81:3271-3276; Bock et al., Nature, (1992) 355: 564-566).

Oligonucleotides and their analogs have been developed and used fordiagnostic purposes, therapeutic applications and as research reagents.For use as therapeutics, oligonucleotides must be transported acrosscell membranes or be taken up by cells, and appropriately hybridize totarget DNA or RNA. These critical functions depend on the initialstability of the oligonucleotides toward nuclease degradation. A seriousdeficiency of unmodified oligonucleotides which affects theirhybridization potential with target DNA or RNA for therapeutic purposesis the enzymatic degradation of administered oligonucleotides by avariety of intracellular and extracellular ubiquitous nucleolyticenzymes referred to as nucleases. For oligonucleotides to be useful astherapeutics or diagnostics, the oligonucleotides should demonstrateenhanced binding affinity to complementary target nucleic acids, andpreferably be reasonably stable to nucleases and resist degradation. Fora non-cellular use such as a research reagent, oligonucleotides need notnecessarily possess nuclease stability.

A number of chemical modifications have been introduced intooligonucleotides to increase their binding affinity to target DNA or RNAand resist nuclease degradation.

Modifications have been made to the ribose phosphate backbone toincrease the resistance to nucleases. These modifications include use oflinkages such as methyl phosphonates, phosphorothioates andphosphorodithioates, and the use of modified sugar moieties such as2′-O-alkyl ribose. Other oligonucleotide modifications include thosemade to modulate uptake and cellular distribution. A number ofmodifications that dramatic alter the nature of the internucleotidelinkage have also been reported in the literature. These includenon-phosphorus linkages, peptide nucleic acids (PNA's) and 2′-5′linkages. Another modification to oligonucleotides, usually fordiagnostic and research applications, is labeling with non-isotopiclabels, e.g., fluorescein, biotin, digoxigenin, alkaline phosphatase, orother reporter molecules.

A variety of modified phosphorus-containing linkages have been studiedas replacements for the natural, readily cleaved phosphodiester linkagein oligonucleotides. In general, most of them, such as thephosphorothioate, phosphoramidates, phosphonates and phosphorodithioatesall result in oligonucleotides with reduced binding to complementarytargets and decreased hybrid stability. In order to make effectivetherapeutics therefore this binding and hybrid stability of antisenseoligonucleotides needs to be improved.

Of the large number of modifications made and studied, few haveprogressed far enough through discovery and development to deserveclinical evaluation. Reasons underlying this include difficulty ofsynthesis, poor binding to target nucleic acids, lack of specificity forthe target nucleic acid, poor in vitro and in vivo stability tonucleases, and poor pharmacokinetics. Several phosphorothioateoligonucleotides and derivatives are presently being used as antisenseagents in human clinical trials for the treatment of various diseasestates. A submission for approval was recently made to both UnitedStates and European regulatory agencies for one antisense drug,Fomivirsen, for use to treat cytomegalovirus (CMV) retinitis in humans.

The structure and stability of chemically modified nucleic acids is ofgreat importance to the design of antisense oligonucleotides. Over thelast ten years, a variety of synthetic modifications have been proposedto increase nuclease resistance, or to enhance the affinity of theantisense strand for its target mRNA (Crooke et al., Med. Res. Rev.,1996, 16, 319-344; De Mesmaeker et al., Acc. Chem. Res., 1995, 28,366-374). Although a great deal of information has been collected aboutthe types of modifications that improve duplex formation, little isknown about the structural basis for the improved affinity observed.

RNA exists in what has been termed “A Form” geometry while DNA exists in“B Form” geometry. In general, RNA:RNA duplexes are more stable, or havehigher melting temperatures (T_(m)) than DNA:DNA duplexes (Sanger etal., Principles of Nucleic Acid Structure, 1984, Springer-Verlag; NewYork, N.Y.; Lesnik et al., Biochemistry, 1995, 34, 10807-10815; Conte etal., Nucleic Acids Res., 1997, 25, 2627-2634). The increased stabilityof RNA has been attributed to several structural features, most notablythe improved base stacking interactions that result from an A-formgeometry (Searle et al., Nucleic Acids Res., 1993, 21, 2051-2056). Thepresence of the 2′ hydroxyl in RNA biases the sugar toward a C3′ endopucker, i.e., also designated as Northern pucker, which causes theduplex to favor the A-form geometry. On the other hand, deoxy nucleicacids prefer a C2′ endo sugar pucker, i.e., also known as Southernpucker, which is thought to impart a less stable B-form geometry(Sanger, W. (1984) Principles of Nucleic Acid Structure,Springer-Verlag, New York, N.Y.). In addition, the 2′ hydroxyl groups ofRNA can form a network of water mediated hydrogen bonds that helpstabilize the RNA duplex (Egli et al., Biochemistry, 1996, 35,8489-8494).

DNA:RNA hybrid duplexes, however, are usually less stable than pureRNA:RNA duplexes, and depending on their sequence may be either more orless stable than DNA:DNA duplexes (Searle et al., Nucleic Acids Res.,1993, 21, 2051-2056). The structure of a hybrid duplex is intermediatebetween A- and B-form geometries, which may result in poor stackinginteractions (Lane et al., Eur. J. Biochem., 1993, 215, 297-306;Fedoroff et al., J. Mol. Biol., 1993, 233, 509-523; Gonzalez et al.,Biochemistry, 1995, 34, 4969-4982; Horton et al., J. Mol. Biol., 1996,264, 521-533). The stability of a DNA:RNA hybrid is central to antisensetherapies as the mechanism requires the binding of a modified DNA strandto a mRNA strand. To effectively inhibit the mRNA, the antisense DNAshould have a very high binding affinity with the mRNA. Otherwise thedesired interaction between the DNA and target mRNA strand will occurinfrequently, thereby decreasing the efficacy of the antisenseoligonucleotide.

One synthetic 2′-modification that imparts increased nuclease resistanceand a very high binding affinity to nucleotides is the 2′-methoxyethoxy(MOE, 2′-OCH₂CH₂OCH₃) side chain (Baker et al., J. Biol. Chem., 1997,272, 11944-12000; Freier et al., Nucleic Acids Res., 1997, 25,4429-4443). One of the immediate advantages of the MOE substitution isthe improvement in binding affinity, which is greater than many similar2′ modifications such as O-methyl, O-propyl, and O-aminopropyl (Freierand Altmann, Nucleic Acids Research, (1997) 25:4429-4443).2′-O-Methoxyethyl-substituted also have been shown to be antisenseinhibitors of gene expression with promising features for in vivo use(Martin, Helv. Chim. Acta, 1995, 78, 486-504; Altmann et al., Chimia,1996, 50, 168-176; Altmann et al., Biochem. Soc. Trans., 1996, 24,630-637; and Altmann et al., Nucleosides Nucleotides, 1997, 16,917-926). Relative to DNA, they display improved RNA affinity and highernuclease resistance. Chimeric oligonucleotides with2′-O-methoxyethyl-ribonucleoside wings and a centralDNA-phosphorothioate window also have been shown to effectively reducethe growth of tumors in animal models at low doses. MOE substitutedoligonucleotides have shown outstanding promise as antisense agents inseveral disease states. One such MOE-substituted oligonucleotide iscurrently available for the treatment of CMV retinitis.

Although the known modifications to oligonucleotides, including the useof the 2′-O-methoxyethyl modification, have contributed to thedevelopment of oligonuclotides for use in diagnostics, therapeutics andas research reagents, there still exists a need in the art for furthermodifications to oligonucleotides having enhanced hybrid bindingaffinity and/or increased nuclease resistance.

SUMMARY OF THE INVENTION

In accordance with the present invention, oligomers containingguanidinium groups are provided. The present invention provides monomersof the formula:

wherein:

-   -   Bx is a heterocyclic base;    -   T₁ is OH or a protected hydroxyl group;    -   T₂ is an activated phosphorus group or a linking moiety attached        to a solid support;    -   T₃ is H, OH, a protected hydroxyl or a sugar substituent group;        said monomer further comprising at least one group, R₁, therein;        said R₁ group occurring in lieu of at least one T₁, T₂ or T₃ or        as a substituent on at least one Bx; said R₁ group having the        formula:        wherein:    -   each Z is, independently, a single bond, O, N or S;    -   each R₂, R₃, R_(3′), and R₄ is, independently, hydrogen, C(O)R₅,        substituted or unsubstituted C₁-C₁₀ alkyl, substituted or        unsubstituted C₂-C₁₀ alkenyl, substituted or unsubstituted        C₂-C₁₀ alkynyl, alkylsulfonyl, arylsulfonyl, a chemical        functional group or a conjugate group, wherein the substituent        groups are selected from hydroxyl, amino, alkoxy, carboxy,        benzyl, phenyl, nitro, thiol, thioalkoxy, halogen, alkyl, aryl,        alkenyl and alkynyl;    -   or R₃ and R₄, together, are R₇;    -   each R₅ is, independently, substituted or unsubstituted C₁-C₁₀        alkyl, trifluoromethyl, cyanoethyloxy, methoxy, ethoxy,        t-butoxy, allyloxy, 9-fluorenylmethoxy,        2-(trimethylsilyl)-ethoxy, 2,2,2-trichloroethoxy, benzyloxy,        butyryl, iso-butyryl, phenyl or aryl;    -   each R₇ is, independently, hydrogen or forms a phthalimide        moiety with the nitrogen atom to which it is attached;    -   each m is, independently, zero or 1; and    -   each n is, independently, an integer from 1 to about 6.

Preferred compositions include oligomers comprising a plurality ofnucleotide units of the structure:

wherein:

-   -   Bx is a heterocyclic base;    -   each T₁, and T₂ is, independently, OH, a protected hydroxyl, a        nucleotide, a nucleoside or an oligonucleotide;    -   T₃ is H, OH, a protected hydroxyl or a sugar substituent group;        said oligomer further comprising at least one group, R₁,        therein; said R₁ group occurring at the 3′ end, the 5′ end, in        lieu of at least one T₃ or as a substituent on at least one Bx;        said R₁ group having the formula:        wherein:    -   each Z is, independently, a single bond, O, N or S;    -   each R₂, R₃, R_(3′), and R₄ is, independently, hydrogen, C(O)R₅,        substituted or unsubstituted C₁-C₁₀ alkyl, substituted or        unsubstituted C₂-C₁₀ alkenyl, substituted or unsubstituted        C₂-C₁₀ alkynyl, alkylsulfonyl, arylsulfonyl, a chemical        functional group or a conjugate group, wherein the substituent        groups are selected from hydroxyl, amino, alkoxy, carboxy,        benzyl, phenyl, nitro, thiol, thioalkoxy, halogen, alkyl, aryl,        alkenyl and alkynyl;    -   or R₃ and R₄, together, are R₇;    -   each R₅ is, independently, substituted or unsubstituted C₁-C₁₀        alkyl, trifluoromethyl, cyanoethyloxy, methoxy, ethoxy,        t-butoxy, allyloxy, 9-fluorenylmethoxy,        2-(trimethylsilyl)-ethoxy, 2,2,2-trichloroethoxy, benzyloxy,        butyryl, iso-butyryl, phenyl or aryl;    -   each R₇ is, independently, hydrogen or forms a phthalimide        moiety with the nitrogen atom to which it is attached;    -   each m is, independently, zero or 1; and    -   each n is, independently, an integer from 1 to about 6.

In a preferred embodiment, R₁, R₂, R₃, R_(3′), and R₄ are hydrogen. Inanother preferred embodiment, R₁, R₂, R₃, R_(3′), and R₄ are hydrogen, mis zero and n is 2.

The present invention also provides methods for preparing oligomerscomprising the steps of:

-   -   (a) selecting a monomer of the formula:        wherein:    -   Bx is a heterocyclic base;    -   T₁ is OH or a protected hydroxyl group;    -   T₂ is a linking moiety attached to a solid support;    -   T₃ is H, OH, a protected hydroxyl or a sugar substituent group;        said monomer further comprising at least one group, R₁, therein;        said R₁ group occurring in lieu of at least one T₁, T₂ or T₃ or        as a substituent on at least one Bx; provided that if T₂ is R₁,        T₃ is a linking moiety attached to a solid support;        said R₁ group having the formula:        wherein:    -   each Z is, independently, a single bond, O, N or S;    -   each R₂, R₃, R_(3′), and R₄ is, independently, hydrogen, C(O)R₅,        substituted or unsubstituted C₁-C₁₀ alkyl, substituted or        unsubstituted C₂-C₁₀ alkenyl, substituted or unsubstituted        C₂-C₁₀ alkynyl, alkylsulfonyl, arylsulfonyl, a chemical        functional group or a conjugate group, wherein the substituent        groups are selected from hydroxyl, amino, alkoxy, carboxy,        benzyl, phenyl, nitro, thiol, thioalkoxy, halogen, alkyl, aryl,        alkenyl and alkynyl;    -   or R₃ and R₄, together, are R₇;    -   each R₅ is, independently, substituted or unsubstituted C₁-C₁₀        alkyl, trifluoromethyl, cyanoethyloxy, methoxy, ethoxy,        t-butoxy, allyloxy, 9-fluorenylmethoxy,        2-(trimethylsilyl)-ethoxy, 2,2,2-trichloroethoxy, benzyloxy,        butyryl, iso-butyryl, phenyl or aryl;    -   each R₇ is, independently, hydrogen or forms a phthalimide        moiety with the nitrogen atom to which it is attached;    -   each m is, independently, zero or 1; and    -   each n is, independently, an integer from 1 to about 6;    -   (b) deprotecting the protected hydroxyl group at the 5′-position        to form a deprotected monomer;    -   (c) coupling said deprotected monomer with a second monomer of        formula:        wherein:    -   Bx is a heterocyclic base;    -   T₁ is OH or a protected hydroxyl group;    -   T₂ is an activated phosphorus group;    -   T₃ is H, OH, a protected hydroxyl or a sugar substituent group;        said monomer further comprising at least one group, R₁, therein;        said R₁ group occurring in lieu of at least one T₁, T₂ or T₃ or        as a substituent on at least one Bx;        provided that if T₂ is R₁, T₃ is an activated phosphorus group;        said R₁ group having the formula:        wherein:    -   each Z is, independently, a single bond, O, N or S;    -   each R₂, R₃, R_(3′), and R₄ is, independently, hydrogen, C(O)R₅,        substituted or unsubstituted C₁-C₁₀ alkyl, substituted or        unsubstituted C₂-C₁₀ alkenyl, substituted or unsubstituted        C₂-C₁₀ alkynyl, alkylsulfonyl, arylsulfonyl, a chemical        functional group or a conjugate group, wherein the substituent        groups are selected from hydroxyl, amino, alkoxy, carboxy,        benzyl, phenyl, nitro, thiol, thioalkoxy, halogen, alkyl, aryl,        alkenyl and alkynyl;    -   or R₃ and R₄, together, are R₇;    -   each R₅ is, independently, substituted or unsubstituted C₁-C₁₀        alkyl, trifluoromethyl, cyanoethyloxy, methoxy, ethoxy,        t-butoxy, allyloxy, 9-fluorenylmethoxy,        2-(trimethylsilyl)-ethoxy, 2,2,2-trichloroethoxy, benzyloxy,        butyryl, iso-butyryl, phenyl or aryl;    -   each R₇ is, independently, hydrogen or forms a phthalimide        moiety with the nitrogen atom to which it is attached;    -   each m is, independently, zero or 1; and    -   each n is, independently, an integer from 1 to about 6, said        coupling occurring in the presence of an activating agent to        form a coupled compound;    -   (d) capping said coupled compound with a capping reagent to form        a capped compound having an internucleotide linkage;    -   (e) oxidizing said internucleotide linkage with an oxidizing        reagent; and    -   (f) repeating steps (b) to (e) to form an oligomer. In one        embodiment of the present invention, the method includes an        additional step of cleaving the oligomer with a cleaving        reagent.

Preferred activating reagents include tetrazole, pyridiniumtrifluoroacetate and dicyanoimidazole. It is preferred that aceticanhydride and N-methylimidazole be the capping reagent. Preferredoxidizing reagents include iodine, camphorsulfonyloxaziridine, t-butylhydrogen peroxide and Beaucage reagent.

The present invention also provides compounds of the formula:

wherein X is cyanoethyloxy, benzyloxy, t-butoxy, methoxy, ethoxy,allyloxy, 9-fluorenylmethoxy, 2-(trimethylsilyl)-ethoxy,2,2,2-trichloroethoxy, trifluoromethyl, butyryl, iso-butyryl, phenyl oraryl.

In one embodiment of the present invention, X is cyanoethyloxy. Inanother embodiment, X is phenyl. In yet another embodiment, X ist-butyl.

The present invention is also directed to non-nucleic acid monomers andoligomers comprising at least one such non-nucleic acid monomer. Thepresent invention includes non-nucleic acid monomers of the formula:

wherein:

-   -   X is C₃-C₁₀ alkyl, C₆-C₂₄ aryl, C₆-C₂₄ heteroaryl, C₄-C₂₀        alicyclic, C₄-C₂₀ alicyclic having at least one heteroatom,        nucleoside, nucleotide or oligonucleotide;    -   Y₁ is a hydroxyl protecting group;    -   Y₂ is an activated phosphorus group or a linking moiety attached        to a solid support;    -   each R₂, R₃, R_(3′), and R₄ is, independently, hydrogen, C(O)R₅,        substituted or unsubstituted C₁-C₁₀ alkyl, substituted or        unsubstituted C₂-C₁₀ alkenyl, substituted or unsubstituted        C₂-C₁₀ alkynyl, alkylsulfonyl, arylsulfonyl, a chemical        functional group or a conjugate group, wherein the substituent        groups are selected from hydroxyl, amino, alkoxy, carboxy,        benzyl, phenyl, nitro, thiol, thioalkoxy, halogen, alkyl, aryl,        alkenyl and alkynyl;    -   or R₃ and R₄, together, are R₇;    -   each R₅ is, independently, substituted or unsubstituted C₁-C₁₀        alkyl, trifluoromethyl, cyanoethyloxy, methoxy, ethoxy,        t-butoxy, allyloxy, 9-fluorenylmethoxy,        2-(trimethylsilyl)-ethoxy, 2,2,2-trichloroethoxy, benzyloxy,        butyryl, iso-butyryl, phenyl or aryl;    -   each R₇ is, independently, hydrogen or forms a phthalimide        moiety with the nitrogen atom to which it is attached; and    -   n is an integer from 1 to about 6.

In a preferred embodiment, Y₁ is dimethoxytrityl. In another preferredembodiment, Y₁ is monomethoxytrityl. In yet another preferredembodiment, Y₂ is a phosphoramidite. In a further embodiment, Y₂ is alinking moiety attached to a solid support. It is preferred that Y₂ besuccinyl CPG.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the structure of a 2′-O-[2-(guanidinium)ethyl]modification.

FIG. 2 shows the binding affinity/modification of2′-O-[2-(guanidinium)ethyl] modification in comparison to other known2′-modifications such as 2′-O-MOE (methoxyethyl), 2′-O-DMAOE(dimethylaminooxyethyl), 2′-O-DEAOE(diethylaminooxyethyl), 2′-O-IPMAOE(isopropylmethylaminooxyethyl), 2′-O-imidazoylethyl (IE) and2′-aminopropyl (AP).

FIG. 3 shows binding affinity of 2′-O-[2-(guanidinium)ethyl] modifiedoligonucleotides as a function of position in placement compared to2′-aminopropyl and 2′-O-DMAEOE (dimethylaminoethyloxyethyl).

FIG. 4 shows electropherograms demonstrating the nuclease resistance of2′-o-[2-(guanidinium)ethyl] modified oligonucleotides.

FIG. 5 shows the synthesis of compound 1.

FIG. 6 shows the synthesis of compound 4.

FIG. 7 shows the structures of compounds 5-7.

FIG. 8 shows the synthesis of compounds 8-13.

FIG. 9 shows the synthesis of compounds 15-18.

FIG. 10 shows the synthesis of compounds 20-21.

FIG. 11 shows the synthesis of compounds 22-23.

FIG. 12 shows the synthesis of compounds 24-25.

FIG. 13 shows the synthesis of compounds 26-29.

FIG. 14 shows the synthesis of compounds 30-33.

FIG. 15 shows the synthesis of compound 34.

FIG. 16 shows the synthesis of compound 35.

FIG. 17 shows the synthesis of compound 36.

FIG. 18 shows the synthesis of compounds 40 and 41.

FIG. 19 shows the synthesis of compound 45.

FIG. 20 shows the synthesis of compound 49.

FIG. 21 shows the synthesis of compound 53.

FIG. 22 shows the synthesis of compound 56.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS

The present invention provides monomers comprising a guanidiniumfunctionality located at the 2′-position or as a substituent on theheterocyclic base. The present invention also provides oligomerscontaining a plurality of nucleotide units, at least one of saidnucleotide units bearing a guanidinium group at the 3′ end, the 5′ end,the 2′-position, or as a substituent on the heterocyclic base. Alsoprovided are methods of making such oligomers.

As used herein, “guanidinium group” or “guanidinium functionality”denotes a group of formula:

wherein:

-   -   Z is a single bond, O, N or S;    -   each R₂, R₃, R_(3′), and R₄ is, independently, hydrogen, C(O)R₅,        substituted or unsubstituted C₁-C₁₀ alkyl, substituted or        unsubstituted C₂-C₁₀ alkenyl, substituted or unsubstituted        C₂-C₁₀ alkynyl, alkylsulfonyl, arylsulfonyl, a chemical        functional group or a conjugate group, wherein the substituent        groups are selected from hydroxyl, amino, alkoxy, carboxy,        benzyl, phenyl, nitro, thiol, thioalkoxy, halogen, alkyl, aryl,        alkenyl and alkynyl;    -   or R₃ and R₄, together, are R₇;    -   each R₅ is, independently, substituted or unsubstituted C₁-C₁₀        alkyl, trifluoromethyl, cyanoethyloxy, methoxy, ethoxy,        t-butoxy, allyloxy, 9-fluorenylmethoxy,        2-(trimethylsilyl)-ethoxy, 2,2,2-trichloroethoxy, benzyloxy,        butyryl, iso-butyryl, phenyl or aryl;    -   each R₇ is, independently, hydrogen or forms a phthalimide        moiety with the nitrogen atom to which it is attached;    -   m is zero or 1; and    -   n is an integer from 1 to about 6.

In one embodiment of the present invention, Z is O, R₂, R₃, R_(3′), andR₄ are hydrogen, n is 1 and m is zero. This 2-O-guanidiniumethylmodification, present in an oligomer, significantly increases thebinding affinity of the oligomer to the target. This increase in bindingaffinity improves when additional guanidinium modifications aredispersed within the oligomer. Guanidinium functionalized oligomers areof use in forming triple helices with double-stranded nucleic acidsmoieties.

The guanidinium group is strongly basic with a pk_(a) of about 12.5, andis a well-stabilized cation with the positive charge being delocalizedover four atoms. Guanidinium groups are understood to facilitate contactbetween proteins and peptides with phosphate groups of nucleic acids.Particularly, the guanidinium group is suggested to form ionicinteractions with guanine residues in the major groove or bond withphosphate moieties located in the minor groove.

The present invention includes oligomers containing nucleotide monomersas well as non-nucleic acid monomers. For example, the present inventionincludes compounds of the following formulas, as well as oligomershaving at least one monomer compound of the following formulas:

wherein each R₆ and R_(6′) is, independently, trifluoromethyl,cyanoethyloxy, methoxy, ethoxy, t-butoxy, allyloxy, 9-fluorenylmethoxy,2-(trimethylsilyl)-ethoxy, 2,2,2-trichloroethoxy, benzyloxy, butyryl,iso-butyryl, phenyl or aryl.

Heterocyclic bases amenable to the present invention include bothnaturally- and non-naturally-occurring nucleobases and heterocycles. Theheterocyclic base may be a pyrimidine, purine or diaminopurine base. Arepresentative list includes adenine, guanine, cytosine, uridine, andthymine, as well as other synthetic and natural nucleobases such asxanthine, hypoxanthine, 2-aminoadenine, 6-methyl and other alkylderivatives of adenine and guanine, 2-propyl and other alkyl derivativesof adenine and guanine, 5-halo uracil and cytosine, 6-azo uracil,cytosine and thymine, 5-uracil (pseudo uracil), 4-thiouracil, 8-halo,oxa, amino, thiol, thioalkyl, hydroxyl and other 8-substituted adeninesand guanines, 7-methylguanine, 5-trifluoromethyl and other 5-substituteduracils and cytosines. Further heterocyclic bases include thosedisclosed in U.S. Pat. No. 3,687,808; the Concise Encyclopedia OfPolymer Science And Engineering, pages 858-859, Kroschwitz, J. I., Ed.,John Wiley & Sons, 1990; and Englisch et al., Angewandte Chemie,International Edition, 1991, 30, 613.

Heterocyclic bases in the oligomers of the present invention may becovalently bound to an R₁ group. In a preferred embodiment, the R₁ groupmay be covalently attached to C5 or an amino group at the 4-position ofa pyrimidine heterocyclic base. In another preferred embodiment, the R₁group may be covalently attached to an amino group at the 2-position or6-position of a purine heterocyclic base. The R₁ group may even beattached to the amino group at the 6-position of a diaminopurineheterocyclic base.

The monomers of the present invention can include appropriate activatedphosphorus groups such as activated phosphate groups and activatedphosphite groups. As used herein, the terms activated phosphate andactivated phosphite groups refer to activated monomers or oligomers thatare reactive with a hydroxyl group of another monomeric or oligomericcompound to form a phosphorus-containing internucleotide linkage. Suchactivated phosphorus groups contain activated phosphorus atoms inP^(III) or P^(V) valency states. Such activated phosphorus atoms areknown in the art and include, but are not limited to, phosphoramidite,H-phosphonate and phosphate triesters. A preferred synthetic solid phasesynthesis utilizes phosphoramidites as activated phosphates. Thephosphoramidites utilize P^(III) chemistry. The intermediate phosphitecompounds are subsequently oxidized to the P^(V) state using knownmethods to yield, in a preferred embodiment, phosphodiester orphosphorothioate internucleotide linkages. Additional activatedphosphates and phosphites are disclosed in Tetrahedron Report Number 309(Beaucage and Iyer, Tetrahedron, 1992, 48, 2223-2311).

In the context of the present invention, the term “oligonucleotide”refers to an oligomer or polymer of ribonucleic acid (RNA) ordeoxyribonucleic acid (DNA) or mimics thereof. This term includesoligonucleotides composed of naturally-occurring nucleobases, sugars andcovalent internucleoside (backbone) linkages as well as oligonucleotideshaving non-naturally-occurring portions which function similarly. Suchmodified or substituted oligonucleotides are often preferred over nativeforms because of desirable properties such as, for example, enhancedcellular uptake, enhanced affinity for nucleic acid target and increasedstability in the presence of nucleases.

Although antisense oligonucleotides are a preferred form of antisensecompounds, the present invention comprehends other oligomeric antisensecompounds, including but not limited to oligonucleotide mimics. Theoligomers in accordance with this invention preferably comprise fromabout 8 to about 30 nucleobases (i.e., from about 8 to about 30 linkednucleosides). Particularly preferred oligomers are antisenseoligonucleotides, even more preferably those comprising from about 12 toabout 25 nucleobases. As is known in the art, a nucleoside is abase-sugar combination. The base portion of the nucleoside is normally aheterocyclic base. The two most common classes of such heterocyclicbases are the purines and the pyrimidines. Nucleotides are nucleosidesthat further include a phosphate group covalently linked to the sugarportion of the nucleoside. For those nucleosides that include apentofuranosyl sugar, the phosphate group can be linked to either the2′, 3′ or 5′ hydroxyl moiety of the sugar. In forming oligonucleotides,the phosphate groups covalently link adjacent nucleosides to one anotherto form a linear polymeric compound. In turn, the respective ends ofthis linear polymeric structure can be further joined to form a circularstructure. Open linear structures are generally preferred. Within theoligonucleotide structure, the phosphate groups are commonly referred toas forming the internucleotide backbone of the oligonucleotide. Thenormal linkage or backbone of RNA and DNA is a 3′ to 5′ phosphodiesterlinkage.

Specific examples of preferred antisense compounds useful in thisinvention include oligonucleotides containing modified backbones ornon-natural internucleotide linkages. As defined in this specification,oligonucleotides having modified backbones include those that retain aphosphorus atom in the backbone and those that do not have a phosphorusatom in the backbone. In the context of the present invention, and assometimes referenced in the art, modified oligonucleotides that do nothave a phosphorus atom in the internucleotide backbone can also beconsidered to be oligonucleosides.

Preferred modified oligonucleotide backbones include, for example,phosphorothioates, chiral phosphorothioates, phosphorodithioates,phosphotriesters, aminoalkylphosphotri-esters, methyl and other alkylphosphonates including 3′-alkylenephosphonates and chiral phosphonates,phosphinates, phosphoramidates including 3′-aminophosphoramidates andaminoalkylphosphoramidates, thionophosphoramidates,thionoalkylphosphonates, thionoalkylphosphotriesters, andboranophosphates having normal 3′-5′ linkages, 2′-5′ linked analogs ofthese, and those having inverted polarity wherein the adjacent pairs ofnucleoside units are linked 3′-5′ to 5′-3′, or 2′-5′ to 5′-2′. Varioussalts, mixed salts and free acid forms are also included.

Representative United States patents that teach the preparation of theabove phosphorus-containing linkages include, but are not limited to,U.S. Pat. Nos. 3,687,808; 4,469,863; 4,476,301; 5,023,243; 5,177,196;5,188,897; 5,264,423; 5,276,019; 5,278,302; 5,286,717; 5,321,131;5,399,676; 5,405,939; 5,453,496; 5,455,233; 5,466,677; 5,476,925;5,519,126; 5,536,821; 5,541,306; 5,550,111; 5,563,253; 5,571,799;5,587,361; and 5,625,050, certain of which are commonly owned with thisapplication.

Preferred modified oligonucleotide backbones that do not include aphosphorus atom therein have backbones that are formed by short chainalkyl or cycloalkyl internucleoside linkages, mixed heteroatom and alkylor cycloalkyl internucleoside linkages, or one or more short chainheteroatomic or heterocyclic internucleoside linkages. These linkagesinclude morpholino linkages (formed in part from the sugar portion of anucleoside); siloxane backbones; sulfide, sulfoxide and sulfonebackbones; formacetyl and thioformacetyl backbones; methylene formacetyland thioformacetyl backbones; alkene containing backbones; sulfamatebackbones; methyleneimino and methylenehydrazino backbones; sulfonateand sulfonamide backbones; amide backbones; and others having mixed N,O, S and CH₂ component parts.

Representative United States patents that teach the preparation of theabove oligonucleosides include, but are not limited to, U.S. Pat. Nos.5,034,506; 5,166,315; 5,185,444; 5,214,134; 5,216,141; 5,235,033;5,264,562; 5,264,564; 5,405,938; 5,434,257; 5,466,677; 5,470,967;5,489,677; 5,541,307; 5,561,225; 5,596,086; 5,602,240; 5,610,289;5,602,240; 5,608,046; 5,610,289; 5,618,704; 5,623,070; 5,663,312;5,633,360; 5,677,437; and 5,677,439, certain of which are commonly ownedwith this application.

In other preferred oligonucleotide mimics, both the sugar and theinternucleoside linkage, i.e., the backbone of the nucleotide units, arereplaced with novel groups. The base units are maintained forhybridization with an appropriate nucleic acid target compound. One sucholigomeric compound, an oligonucleotide mimic that has been shown tohave excellent hybridization properties, is referred to as a peptidenucleic acid (PNA). In PNA compounds, the sugar-backbone of anoligonucleotide is replaced with an amide-containing backbone, inparticular an aminoethylglycine backbone. The nucleobases are retainedand are bound directly or indirectly to aza nitrogen atoms of the amideportion of the backbone. Representative United States patents that teachthe preparation of PNA compounds include, but are not limited to, U.S.Pat. Nos. 5,539,082; 5,714,331; and 5,719,262. Further teachings of PNAcompounds can be found in Nielsen et al. (Science, 1991, 254,1497-1500).

In one embodiment of the present invention, oligonucleotides withphosphorothioate backbones are preferred. Also preferred areoligonucleosides with heteroatom backbones, and in particular—CH₂—NH—O—CH₂—, —CH₂—N(CH₃)—O—CH₂— [known as a methylene (methylimino)or MMI backbone], —CH₂—O—N(CH₃)—CH₂—, —CH₂—N(CH₃)—N(CH₃)—CH₂— and—O—N(CH₃)—CH₂—CH₂— [wherein the native phosphodiester backbone isrepresented as —O—P—O—CH₂—] of the above referenced U.S. Pat. No.5,489,677, and the amide backbones of the above referenced U.S. Pat. No.5,602,240. Also preferred are oligonucleotides having morpholinobackbone structures of the above-referenced U.S. Pat. No. 5,034,506.

The methods according to the present invention are performed in varioustraditional solvents either utilizing solution phase techniques orautomated synthetic protocols. Many solvents for automatedoligonucleotide synthesis as well as solution phase oligonucleotidesynthesis are known in the art. Preferred solvents include DMF, DMSO,THF, THP and CH₃CN.

Standard solution phase and solid phase methods for the synthesis ofoligonucleotides and oligonucleotide analogs are well known to thoseskilled in the art. These methods are constantly being improved in waysthat reduce the time and cost required to synthesize these complicatedcompounds. Representative solution phase techniques are described inU.S. Pat. No. 5,210,264, issued May 11, 1993 and commonly assigned withthis invention. Representative solid phase techniques employed foroligonucleotide and oligonucleotide analog synthesis utilizing standardphosphoramidite chemistries are described in “Protocols ForOligonucleotides And Analogs,” Agrawal, S., Ed., Humana Press, Totowa,N.J., 1993.

A preferred method of choice for the preparation of naturally-occurringoligonucleotides, as well as non-naturally-occurring (or modified)oligonucleotides such as phosphorothioate oligonucleotides, is viasolid-phase synthesis wherein an oligonucleotide is prepared on apolymer support (a solid support) such as controlled pore glass (CPG);oxalyl-controlled pore glass (see, e.g., Alul et al., Nucleic AcidsResearch 1991, 19, 1527); TENTAGEL Support, (see, e.g., Wright et al.,Tetrahedron Letters 1993, 34, 3373); or POROS, a polystyrene resinavailable from PerSeptive Biosystems. Equipment for such synthesis iscommercially available from several vendors including, for example,Applied Biosystems (Foster City, Calif.). Any other means for suchsynthesis known in the art may additionally or alternatively beemployed. Suitable solid phase techniques, including automated synthesistechniques, are described in F. Eckstein (ed.), Oligonucleotides andAnalogues, a Practical Approach, Oxford University Press, New York(1991).

Solid-phase synthesis relies on sequential addition of nucleotides toone end of a growing oligonucleotide chain. Typically, a firstnucleoside (having protecting groups on any exocyclic aminefunctionalities present) is attached to an appropriate glass beadsupport. Activated phosphite compounds (typically nucleotidephosphoramidites, also bearing appropriate protecting groups) are addedstepwise to elongate the growing oligonucleotide. Additional methods forsolid-phase synthesis may be found in U.S. Pat. Nos. 4,415,732;4,458,066; 4,500,707; 4,668,777; 4,973,679; 5,132,418; 4,725,677 and Re.34,069.

A representative list of chemical functional groups according to theinvention include C₁-C₁₀ alkyl, substituted C₁-C₁₀ alkyl, C₂-C₂₅alkenyl, substituted C₂-C₂₅ alkenyl, C₂-C₁₅ alkynyl, substituted C₂-C₁₅alkynyl, C₄-C₇ carbocyclic alkyl, substituted carbocyclic alkyl, alkenylcarbocyclic, substituted alkenyl carbocyclic, alkynyl carbocyclic,substituted alkynyl carbocyclic, C₆-C₂₀ aryl, substituted C₆-C₂₀ aryl,heteroaryl, substituted heteroaryl, a nitrogen, oxygen, or sulfurcontaining heterocycle, a substituted nitrogen, oxygen, or sulfurcontaining heterocycle, a mixed heterocycle, or a substituted mixedheterocycle, where said substituent groups are selected from alkyl,alkenyl, alkynyl, aryl, hydroxyl, amino, alkoxy, carboxy, benzyl, nitro,thiol, thioalkyl, thioalkoxy, or halogen groups; or L is phthalimido, anether having 2 to 10 carbon atoms and 1 to 4 oxygen or sulfur atoms, ametal coordination group, a conjugate group, halogen, hydroxyl, thiol,keto, carboxyl, NR¹R², CONR¹, amidine (C(═NH)NR²R³), guanidine(NHC(═NH)NR²R³), glutamyl (R₁OOCCH(NR²R³) (CH₂)₂C(═O), nitrate, nitro,nitrile, trifluoromethyl, trifluoromethoxy, NH-alkyl, N-dialkyl,O-aralkyl, S-aralkyl, NH-aralkyl, azido (N₃), hydrazino (NHNH₂),hydroxylamino (ONH₂), sulfoxide (SO), sulfone (SO₂), sulfide (S—),disulfide (S—S), silyl, a nucleosidic base, an amino acid side chain, acarbohydrate, a drug, or a group capable of hydrogen bonding, whereineach R¹ and R² is, independently, H, haloalkyl, C₁-C₁₀ alkyl, C₂-C₁₀alkenyl, C₂-C₁₀ alkynyl, or C₆-C₁₄ aryl; and each R³ is, independently,a single bond, CH═CH, C≡C, O, S, NR⁶, SO₂, C₆-C₁₄ aryl, substitutedC₆-C₁₄ aryl, heteroaryl, substituted heteroaryl, a nitrogen, oxygen, orsulfur containing heterocycle, a substituted nitrogen, oxygen, or sulfurcontaining heterocycle, a mixed heterocycle, or a substituted mixedheterocycle, wherein said substituent groups are selected from hydroxyl(OH), amino, alkoxy, carboxy, benzyl, phenyl, nitro (NO₂), thiol (SH),thioalkoxy, halogen, alkyl, aryl, alkenyl, and alkynyl groups.

A number of chemical functional groups can be introduced into compoundsof the present invention in a blocked form and can then be subsequentlydeblocked to form the final, desired compound. In general, a blockinggroup renders a chemical functionality of a molecule inert to specificreaction conditions and can later be removed from such functionality ina molecule without substantially damaging the remainder of the molecule(Green and Wuts, Protective Groups in Organic Synthesis, 2d edition,John Wiley & Sons, New York, 1991). For example, amino groups can beblocked as phthalimido, 9-fluorenylmethoxycarbonyl (FMOC),triphenylmethylsulfenyl, t-BOC or benzyl groups. Carboxyl groups can beprotected as acetyl groups.

Representative hydroxyl protecting groups are described by Beaucage etal. (Tetrahedron 1992, 48, 2223). Preferred hydroxyl protecting groupsare acid-labile, such as trityl, monomethoxytrityl, dimethoxytrityl,trimethoxytrityl, 9-phenylxanthine-9-yl (Pixyl) and9-(ρ-methoxyphenyl)-xanthine-9-yl (MOX). Chemical functional groups canalso be “blocked” by including them in a precursor form. Thus an azidogroup can be considered to be a “blocked” form of an amine as the azidogroup may be easily converted to the amine. Further representativeprotecting groups utilized in oligonucleotide synthesis are discussed inAgrawal et al., Protocols for Oligonucleotide Conjugates, Humana Press,New Jersey, 1994, Vol. 26, pp. 1-72.

In the context of the present invention, a “heterocycle” is a cycliccompound containing at least one heteroatom such as N, O or S. A “mixedheterocycle” is a cyclic compound containing at least two heteroatomssuch as N, O or S. A “heteroaryl” compound is a heterocycle containingat least one heteroatom such as N, O or S and is not fully saturated,e.g., is in a state of partial or complete saturation. “Heteroaryl” isalso meant to include fused systems including systems where one or moreof the fused rings contain no heteroatoms. Heterocycles, includingnitrogen heterocycles, according to the present invention include, butare not limited to, imidazole, pyrrole, pyrazole, indole, 1H-indazole,α-carboline, carbazole, phenothiazine, phenoxazine, tetrazole, triazole,pyrrolidine, piperidine, piperazine and morpholine groups. A morepreferred group of nitrogen heterocycles includes imidazole, pyrrole,indole, and carbazole groups.

As used herein, “linking moiety” refers to a hydrocarbyl chain whichconnects the monomers and oligomers of the invention to a solid support.A preferred linking moiety is a succinyl group. Other linking moietiesinclude, but are not limited to, substituted or unsubstituted C₁-C₁₀alkyl, substituted or unsubstituted C₂-C₁₀ alkenyl or substituted orunsubstituted C₂-C₁₀ alkynyl, wherein the substituent groups areselected from hydroxyl, amino, alkoxy, carboxy, benzyl, phenyl, nitro,thiol, thioalkoxy, halogen, alkyl, aryl, alkenyl and alkynyl.

“Conjugate groups” according to the present invention include thoseknown in the art. A representative list of conjugate groups amenable tothe present invention includes intercalators, reporter molecules,contrast reagents, cleaving agents, cell targeting agents, cyanine dyes,polyamines, polyamides, poly ethers including polyethylene glycols, andother moieties known in the art for enhancing the pharmacodynamicproperties or the pharmacokinetic properties. Typical conjugate groupsinclude PEG groups, cholesterols, phospho-lipids, biotin,phenanthroline, phenazine, pyrene, retinal, phenanthridine,anthraquinone, acridine, fluoresceins, rhodamines, coumarins, and dyes.

For the purposes of this invention, the term “reporter molecule”includes molecules or enzymes that have physical or chemical propertiesthat allow them to be identified in gels, fluids, whole cellularsystems, broken cellular systems and the like utilizing physicalproperties such as spectroscopy, radioactivity, colorimetric assays,fluorescence, and specific binding. Particularly useful as reportermolecules are fluorophores, chromophores and radiolabel-containingmoieties.

Fluorophores are molecules detectable by fluorescence spectroscopy.Examples of preferred fluorophores are fluorescein and rhodamine dyesand acridines. There are numerous commercial available fluorophoresincluding “Texas Red” and other like fluoresceins and rhodaminesavailable from Molecular Probes, Eugene, Oreg.

Chromophores are molecules capable of detection by visible orultraviolet (UV-VIS) absorbance spectroscopy. Examples of chromophoresare polynuclear aromatics such as anthracene, perylene, pyrene,rhodamine and chrysene.

Radiolabel-containing moieties, as used herein, are moleculesincorporating at least one radioactive atom, such as ³H or ¹⁴C, enablingdetection thereby.

Reporter enzymes may be detected directly or via their enzymaticproducts by any of the methods mentioned above. Particularly useful asreporter enzymes are alkaline phosphatase and horseradish peroxidase.

Intercalators are polycyclic aromatic moieties that can insert betweenadjacent base pairs without affecting normal Watson-Crick base pairing,and include hybrid intercalator/ligands such as thephotonuclease/intercalator ligand6-[[[9-[[6-(4-nitrobenzamido)hexyl]amino]acridin-4-yl]carbonyl]amino]hexanoylpentafluorophenylester. This compound has two noteworthy features: an acridine moietythat is an intercalator and a ρ-nitrobenzamido group that is aphotonuclease. Other representative intercalators are disclosed byManoharan, M., Antisense Research and Applications, Crooke and Lebleu,eds., CRC Press, Boca Raton, 1993.

In the context of this invention, “hybridization” means hydrogenbonding, which may be Watson-Crick, Hoogsteen or reversed Hoogsteenhydrogen bonding, between complementary heterocyclic bases.“Complementary,” as used herein, refers to the capacity for precisepairing between two heterocyclic bases. For example, adenine and thymineare complementary bases which pair through the formation of hydrogenbonds. “Complementary” and “specifically hybridizable,” as used herein,refer to precise pairing or sequence complementarity between a first anda second nucleic acid-like oligomer containing nucleoside subunits. Forexample, if a heterocyclic base at a certain position of the firstnucleic acid is capable of hydrogen bonding with a heterocyclic base atthe same position of the second nucleic acid, then the first nucleicacid and the second nucleic acid are considered to be complementary toeach other at that position. The first and second nucleic acids arecomplementary to each other when a sufficient number of correspondingpositions in each molecule are occupied by bases which can hydrogen bondwith each other. Thus, “specifically hybridizable” and “complementary”are terms which are used to indicate a sufficient degree ofcomplementarity such that stable and specific binding occurs between acompound of the invention and a target nucleic acid molecule. It isunderstood that an oligomer of the invention need not be 100%complementary to its target nucleic acid to be specificallyhybridizable. An oligomer is specifically hybridizable when binding ofthe oligomer to the target nucleic acid interferes with the normalfunction of the target to cause a loss of utility, and there is asufficient degree of complementarity to avoid non-specific binding ofthe oligomer to non-target sequences under conditions in which specificbinding is desired, i.e. under physiological conditions in the case ofin vivo assays or therapeutic treatment, or in the case of in vitroassays, under conditions in which the assays are performed.

In the context of the present invention, “modulating” means altering ormodifying, and includes increasing or decreasing. Accordingly,modulating gene expression means increasing or decreasing geneexpression. In one aspect of the invention, modulating gene expressionmeans decreasing gene expression.

As used herein the term “sugar substituent group” or “2′-substituentgroup” includes groups attached to the 2′ position of the ribosyl moietywith or without an oxygen atom. 2′-Sugar modifications amenable to thepresent invention include fluoro, O-alkyl, O-alkylamino, O-alkylalkoxy,protected O-alkylamino, O-alkylaminoalkyl, O-alkyl imidazole, andpolyethers of the formula (O-alkyl)_(m), where m is 1 to about 10.Preferred among these polyethers are linear and cyclic polyethyleneglycols (PEGs), and PEG-containing groups, such as crown ethers, andother reported substituent groups. See, Ouchi et al., Drug Design andDiscovery 1992, 9, 93; Ravasio et al., J. Org. Chem. 1991, 56, 4329; andDelgardo et al., Critical Reviews in Therapeutic Drug Carrier Systems1992, 9, 249. Further sugar substituent groups are disclosed by Cook(Anti-Cancer Drug Design, 1991, 6, 585-607). Fluoro, O-alkyl,O-alkylamino, O-alkyl imidazole, O-alkylaminoalkyl, and alkyl aminosubstituents are described in U.S. Pat. No. 6,166,197, filed Mar. 6,1995, entitled “Oligomeric Compounds having Pyrimidine Nucleotide(s)with 2′ and 5′ Substitutions.”

Additional 2′ sugar modifications amenable to the present inventioninclude 2′-SR and 2′-NR₂ groups, where each R is, independently,hydrogen, a protecting group or substituted or unsubstituted alkyl,alkenyl, or alkynyl. 2′-SR nucleosides are disclosed in U.S. Pat. No.5,670,633, issued Sep. 23, 1997. The incorporation of 2′-SR monomersynthons are disclosed by Hamm et al., J. Org. Chem., 1997, 62,3415-3420. 2′-NR₂ nucleosides are disclosed by Goettinigen, M., J. Org.Chem., 1996, 61, 6273-6281; and Polushin et al., Tetrahedron Lett.,1996, 37, 3227-3230. Further representative 2′-O-sugar modificationsamenable to the present invention include those having one of formula Ior II:

wherein:

-   -   E is C₁-C₁₀ alkyl, N(R₈)(R₉) or N═C(R₈)(R₉);    -   each R₈ and R₉ is, independently, H, C₁-C₁₀ alkyl, a nitrogen        protecting group, or R₈ and R₉, together, are a nitrogen        protecting group or are joined in a ring structure that includes        at least one additional heteroatom selected from N and O;    -   R₁₀ is OX, SX, or N(X)₂;    -   each X is, independently, H, C₁-C₈ alkyl, C₁-C₈ haloalkyl,        C(═NH)N(H)Z, C(═O)N(H)Z or OC(═O)N(H)Z;    -   Z is H or C₁-C₈ alkyl;    -   L₁, L₂ and L₃ comprise a ring system having from about 4 to        about 7 carbon atoms or having from about 3 to about 6 carbon        atoms and 1 or 2 heteroatoms, said heteroatoms being selected        from oxygen, nitrogen and sulfur, wherein said ring system is        aliphatic, unsaturated aliphatic, aromatic, or saturated or        unsaturated heterocyclic;    -   Y is C₁-C₁₀ alkyl or haloalkyl, C₂-C₁₀ alkenyl, C₂-C₁₀ alkynyl,        C₆-C₁₄ aryl, N(R₈) (R₉) OR₈, halo, SR₈ or CN;    -   each q₁ is, independently, an integer from 2 to 10;    -   each q₂ is 0 or 1;    -   p is an integer from 1 to 10; and    -   r is an integer from 1 to 10;        provided that when p is 0, r is greater than 1.

Representative 2′-O-sugar substituents of formula I are disclosed inU.S. Pat. No. 6,172,209, filed Aug. 7, 1998, entitled “Capped2′-Oxyethoxy Oligonucleotides,” hereby incorporated by reference in itsentirety.

Representative cyclic 2′-O— sugar substituents of formula II aredisclosed in U.S. Pat. No. 6,271,356, filed Jul. 27, 1998, entitled “RNATargeted 2′-Modified Oligonucleotides that are ConformationallyPreorganized.”

In the context of the present invention, “alkyl” means substituted orunsubstituted hydrocarbyl groups wherein the carbon atoms are connectedvia single bonds. “Alkenyl” means substituted or unsubstitutedhydrocarbyl moieties having at least one double bond. “Alkynyl” meanssubstituted or unsubstituted hydrocarbyl moieties having at least onetriple bond.

Another modification that is used to prepare oligomeric compoundsamenable to the present invention includes locked nucleic acids (LNA's)which are novel conformationally restricted oligonucleotide analogscontaining 2′-O,4′-C-methylene LNA monomers (see, Singh et al., Chem.Commun., 1998, 4, 455-456). LNA and LNA analogs display very high duplexthermal stabilities with complementary DNA and RNA (Tm=+3 to +10 C),stability towards 3′-exonucleolytic degradation, and good solubilityproperties.

Synthesis of the LNA monomers adenine, cytosine, guanine,5-methylcytosine, thymine and uracil, their oligomerization, and nucleicacid recognition properties have been described (see Koshkin et al.,Tetrahedron, 1998, 54, 3607-3630). Studies of mis-matched sequences showthat LNA obey the Watson-Crick base pairing rules with generallyimproved selectivity compared to the corresponding unmodified referencestrands.

Potent and nontoxic antisense oligonucleotides containing LNA's havebeen described (Wahlestedt et al., Proc. Natl. Acad. Sci. U.S.A., 2000,97, 5633-5638.) The authors have demonstrated that LNA's confer severaldesired properties to antisense agents. LNA/DNA copolymers were notdegraded readily in blood serum and cell extracts. LNA/DNA copolymersexhibited potent antisense activity on assay systems as disparate as aG-protein-coupled receptor in living rat brain and an Escherichia colireporter gene.

The conformations of LNA's has been determined by 2D NMR spectroscopy toshow that the locked conformation of the LNA nucleotides both in ssLNAand in the duplexes organize the phosphate backbone in such a way as tointroduce higher population of the N-type conformation (See, Petersen etal., J. Mol. Recognit., 2000, 13, 44-53). These conformational changesare associated with improved stacking of the nucleobases (see also,Wengel et al., Nucleosides Nucleotides, 1999, 18, 1365-1370).

LNA's form duplexes with complementary DNA, RNA or LNA with high thermalaffinities. CD spectra show that duplexes involving fully modified LNA(esp. LNA:RNA) structurally resemble an A-form RNA:RNA duplex. NMRexamination of an LNA:DNA duplex confirm the 3′-endo conformation of anLNA monomer. Recognition of double-stranded DNA is demonstratedsuggesting strand invasion by LNA. Lipofectin-mediated efficientdelivery of LNA into living human breast cancer cells has beenaccomplished.

Preparation of locked nucleoside analogs-containingoligodeoxyribonucleotide duplexes as substrates for nucleic acidpolymerases has been described (see, Wengel et al., PCT InternationalApplication WO 98-DK393 19980914). The novel type of LNA modifiedoligonucleotides, as well as the LNAs as such, are useful in a widerange of diagnostic applications as well as therapeutic applications.Among these are included antisense applications, PCR applications,strand displacement oligomers, as substrates for nucleic acidpolymerases, and as nucleotide based drugs.

LNA has been shown to form exceedingly stable LNA:LNA Duplexes (Koshkinet al., J. Am. Chem. Soc., 1998, 120, 13252-13253). LNA:LNAhybridization was shown to be the most thermally stable nucleic acidtype duplex system, and the RNA-mimicking character of LNA wasestablished at the duplex level. Introduction of 3 LNA monomers (T or A)induced significantly increase melting points (T=+15/+11) toward DNAcomplements. The universality of LNA-mediated hybridization has beenstressed by the formation of exceedingly stable LNA:LNA duplexes. TheRNA-mimicking of LNA was reflected with regard to the N-typeconformational restriction of the monomers and to the secondarystructure of the LNA:RNA duplex.

Synthesis of 2′-amino-LNA, a novel conformationally restrictedhigh-affinity oligonucleotide analog with a Handle has been shown (seeSingh et al., J. Org. Chem., 1998, 63, 10035-10039.)

2′-Amino- and 2′-methylamino-LNA's were prepared and thermal stabilityof their duplexes with complementary RNA and DNA strands have beenpreviously reported. Similarly, The first analogs of LNA,phosphorothioate-LNA and 2′-thio-LNAs have been prepared (see Kumar etal., Bioorg. Med. Chem. Lett., 1998, 8, 2219-2222.)

As used herein, “alicyclic” means hydrocarbyl groups having a saturatedcyclic structure wherein the carbon atoms are connected by single bonds.Alicyclic groups according to the present invention may also containheteroatoms within the cyclic structure or as part of an exocyclicsubstituent.

Further, in the context of the present invention, “aryl” (generallyC₆-C₂₄) includes, but is not limited to, substituted and unsubstitutedaromatic hydrocarbyl groups. Aralkyl groups (generally C₇-C₂₅) include,but are not limited to, groups having both aryl and alkylfunctionalities, such as benzyl and xylyl groups. Preferred aryl andaralkyl groups include, but are not limited to, phenyl, benzyl, xylyl,naphthyl, toluoyl, pyrenyl, anthracyl, azulyl, phenethyl, cinnamyl,benzhydryl, and mesityl. Typical substituents for substitution include,but are not limited to, hydroxyl, amino, alkoxy, carboxy, benzyl,phenyl, nitro, thiol, thioalkoxy, halogen, alkyl, aryl, alkenyl, oralkynyl groups.

Formulation of therapeutic compositions utilizing compounds of thepresent invention and their subsequent administration is believed to bewithin the skill of those in the art. Dosing is dependent on severityand responsiveness of the disease state to be treated, with the courseof treatment lasting from several days to several months, or until acure is effected or a diminution of the disease state is achieved.Optimal dosing schedules can be calculated from measurements of drugaccumulation in the body of the patient. Persons of ordinary skill caneasily determine optimum dosages, dosing methodologies and repetitionrates. Optimum dosages may vary depending on the relative potency ofindividual oligomers, and can generally be estimated based on EC₅₀sfound to be effective in in vitro and in vivo animal models. In general,dosage is from 0.01 ug to 100 g per kg of body weight, and may be givenonce or more daily, weekly, monthly or yearly, or even once every 2 to20 years. Persons of ordinary skill in the art can easily estimaterepetition rates for dosing based on measured residence times andconcentrations of the drug in bodily fluids or tissues. Followingsuccessful treatment, it may be desirable to have the patient undergomaintenance therapy to prevent the recurrence of the disease state,wherein the oligomer is administered in maintenance doses, ranging from0.01 ug to about 10 g per kg of body weight, once or more daily, to onceevery 20 years.

Additional objects, advantages and novel features of the presentinvention will become apparent to those skilled in the art uponexamination of the following examples. The following examples illustratethe invention and are not intended to limit the same. Those skilled inthe art will recognize, or be able to ascertain through routineexperimentation, numerous equivalents to the specific substances,compositions and procedures described herein. Such equivalents areconsidered to be within the scope of the present invention.

EXAMPLES

All reagents and solvents were purchased from commercial sources unlessotherwise noted. 2-Cyanoethanol, N,N′-disuccinimidyl carbonate (DSC),N-(2-hydroxy)-phthalimide,2-cyanoethyl-N,N,N′,N′-tetraisopropylphosphoro-diamidite,6-amino-hexanol and 2-methyl-2-thiopseudourea-sulfate were obtained fromAldrich Chemical Co., Inc. (Milwaukee, Wis.). Reagents for the DNAsynthesizer were purchased from PerSeptive Biosystems, Inc. (Framingham,Mass.). 2,2′-Anhydro-5-methyluridine was purchased from Ajinomoto(Tokyo, Japan). Flash chromatography was performed on silica gel (Baker,40 mm). Thin-layer chromatography was performed on Kieselgel glassplates from E. Merck and visualized with UV light andp-anisaldehyde/sulfuric acid/acetic acid spray followed by charring.

For all derivatizations described herein, the derivatized material canbe used as obtained without further purification.

Example 1 Synthesis ofN-(2-cyanoethoxycarbonyloxy)succinimide(CEOC-O-Succinimide) (1)

To a stirred solution of 2-cyanoethanol (7.23 g, 102 mmol) in 300 mL ofanhydrous CH₃CN, under argon atmosphere, N,N′-disuccinimidyl carbonate(34.0 g, 133 mmol) was added followed by pyridine (11.3 mL, 140 mmol).The suspension became a clear solution after about 1 h. The solution wasstirred for an additional 6 h and then concentrated in vacuo. Theresidue was redissolved in dichloromethane (200 mL), extracted withsaturated NaHCO₃ solution (3×50 mL) followed by saturated NaCl solution(3×50 mL). The organic layer was dried (anhydrous Na₂SO₄) andconcentrated to afford a white solid. Traces of pyridine were removed byco-evaporation with dry acetonitrile. The white solid was driedovernight in vacuo and then triturated with ether (150 mL) to yield20.23 g (94%) of 1 as a colorless amorphous powder. This material isstable at room temperature in a desiccator for an extended period (1-2years). The proton and carbon NMR spectra revealed a homogeneousmaterial even at this stage. The material was further purified bychromatography on silica gel using CH₂Cl₂:EtOAc (50:50) to give a whitecrystalline compound (18.72 g, 87%); R_(f)=0.21; m.p. 105.5° C.

1H NMR (400 MHz, CDCl₃): 2.85 (t, J=6.62 Hz, 2H), 2.86 (s, 4H), 4.45 (t,J=5.96 Hz); ¹³C NMR (80 MHz, DMSO-d₆): 17.33, 25.39, 65.86, 117.91,150.91, 169.82; HRMS(FAB): Calcd for C₈H₉N₂O₅ ⁺ 213.0511, Found:213.0509; Anal: Calcd for C₈H₈N₂O₅: C, 45.29; H, 3.80; N, 13.20; Found:C, 45.19; H, 3.45; N, 13.02.

Example 2 Synthesis of 2′-O-phthalimidoethyl-5-methyluridine (2)

N-(2-Hydroxyethyl)phthalimide (277 g, 1.45 mol) was slowly added to asolution of borane in tetrahydrofuran (1 M, 600 mL), with stirring.Hydrogen gas evolved as the solid dissolved. Once the rate of gasevolution subsided, the solution was placed in a 2 L stainless steelbomb. 2,2′-Anhydro-5-methyluridine (60 g, 0.25 mol) and sodiumbicarbonate (120 mg) were added and the bomb was sealed. After 30minutes, the bomb was vented and placed in an oil bath and heated to150° C. internal temperature for 24 h. The bomb was cooled to roomtemperature and opened. TLC (ethyl acetate-methanol; 95:5) revealed thedisappearance of starting material. The crude solution was concentratedand the residue was purified by chromatography on silica gel startingwith ethyl acetate to remove the excess phthalimide reagent, followed byethyl acetate-methanol (95:5) to elute 2 (22.2 g, 20.6%).

1H NMR (200 MHz, DMSO-d₆): 1.8 (s, 3H), 3.4-4.2 (m, 6H), 5.0-5.2 (m,2H), 5.8 (d, J=5.1 Hz, 1H), 7.65 (s, 1H), 7.8-8.0 (m, 4H), 11.2 (s, 1H).

Example 3 Synthesis of2′-O-(2-phthalimidoethyl)-5′-O-(4,4′-dimethoxy-trityl)-5-methyl Uridine(3)

2′-O-Phthalimidoethyl-5-methyluridine (2, 22.2 g, 0.053 mol) wascoevaporated with pyridine (2×75 mL) and then dissolved in 100 mL ofpyridine. Dimethoxytrityl chloride (27 g, 0.080 mol) was added in oneportion, with stirring, TLC (ethyl acetate-hexanes 50:50), after 1 h,indicated completion of the reaction. Methanol (10 mL) was added toquench the reaction. The mixture was concentrated and the residue waspartitioned between ethyl acetate and saturated sodium bicarbonatesolution (150 mL each). The organic layer was concentrated and theresidue was dissolved in a minimum amount of dichloromethane and appliedto a silica gel column. The compound was eluted with ethylacetate-hexanes-triethylamine (50:50:1 to 80:20:1) to give 3 (26.1 g,68%) as a white foam.

1H NMR (200 MHz, CDCl₃): 1.33 (s, 3H), 3.05 (d, J=8.4 Hz, 1H), 3.49 (m,2H), 3.8 (s, 6H), 3.9-4.1 (m, 4H), 4.18-4.26 (m, 1H), 4.47 (m, 1H), 5.88(s, 1H), 6.84 (d, J=8.78 Hz, 4H), 7.22-7.43 (m, 9H), 7.69-7.88 (m, 5H),8.26 (s, 1H); HRMS(FAB): Calcd for C₄₁H₃₉N₃O₁₀Na⁺ 756.2533, Found:756.2553.

Example 4 Synthesis of 2′-O-(2-aminoethyl)-5′—O—(4,4′-dimethoxytrityl)-5-methyluridine (4)

2′-O-Phthalimidoethyl-5′-O-DMT-5-methyluridine (3, 21.1 g, 0.029 mol)was dissolved in methanol (500 mL). Anhydrous hydrazine (4.9 mL, 0.15mol) was added and the solution was heated to reflux. TLC after 3 hindicated a complete reaction. The residue was purified bychromatography on silica gel using methanol and then methanol-ammoniumhydroxide (98:2) to give 4 (12.4 g, 70%). The material was completelysoluble in methylene chloride and traces of silica from leaching of thecolumn were removed by filtration at this stage and reevaporating thesolution.

1H NMR (200 MHz, CDCl₃): 1.39 (s, 3H), 2.98 (t, J=3.48 Hz, 2H), 3.45 (d,J=2.56 Hz, 1H), 3.53 (d, J=1.96 Hz, 1H), 3.56-3.68 (m, 2H), 3.81 (s,6H), 3.99 (m, 1H), 4.1 (t, J=4.56 Hz, 1H), 4.17 (m, 1H), 4.45 (t, J=5.06Hz, 1H), 6.06 (d, J=4.12 Hz, 1H), 6.86 (d, J=8.9 Hz, 4H), 7.25-7.46 (m,9H), 7.67 (s, 1H); ¹³C (50 MHz, CDCl₃): 11.71, 40.55, 45.76, 55.03,62.47, 69.15, 70.65, 82.64, 83.49, 86.62, 87.10, 110.98, 113.09, 126.91,127.77, 127.97, 129.95, 135.35, 144.25, 151.27, 158.46, 164.97; HRMS(FAB): Calcd for C₃₃H₃₇O₈N₃Na- 626.2478, Found: 626.2501.

Example 5 Synthesis of N,N′-bis-CEOC-2-methyl-2-thiopseudourea (5)

2-Methyl-2-thiopseudourea.½H₂SO₄ (5.29 g, 38.0 mmol) was suspended inCH₂Cl₂ (250 mL) and saturated NaHCO₃ solution (250 mL).Cyanoethoxycarbonyloxysuccinimide 1 (20.2 g, 95.3 mmol) was added andthe reaction stirred for 2 h. The organic phase was separated. Theaqueous phase was extracted with DCM (2×200 ml) and the combined organicphase was dried (Na₂SO₄), filtered and evaporated. The crude product waspurified by flash chromatography with AcOEt/DCM (95:5) as eluant toafford 5 (3.78 g, 35%) as a white solid.

1H-NMR (200 MHz, CDCl₃): 11.80 (br s, 1H), 4.39 (q, 4H), 2.80 (t, 4H),2.45 (s, 3H).

Example 6 Synthesis of N,N′-bis-benzoyl-2-methyl-2-thiopseudourea (6)

This compound was prepared as described by Derocque et al. (Bulletin dela Société Chimique de France, 1968, 5, 2062-2066).

Example 7 Synthesis of N,N′-bis-pivaloyl-2-methyl-2-thiopseudourea (7)

2-Methyl-2-thiopseudourea.½ H₂SO₄ (1.0 g, 3.60 mmole) was suspended indry pyridine (14.4 ml). Pivaloylchloride (1.77 mL, 14.4 mmole) was addedand the reaction mixture was stirred overnight. The reaction wasquenched by addition of 5% aqueous NaHCO₃ solution (100 mL) andextracted with dichloromethane (2×50 mL). The organic phase was dried(Na₂SO₄), filtered and evaporated. The crude product was purified byflash column chromatography with EtOAc as eluant to afford 7 (1.27 g,68%).

1H-NMR (200 MHz, DMSO-d₆): 13.10 (s, 1H), 2.39 (s, 3H), 1.22 (s, 18H);HRMS (FAB): Calcd for C₁₂H₂₃N₂O₂S- 259.480, Found: 259.1485.

Example 8 Synthesis of 2′-O-[(N,N′-bis-CEOC-guanidinium)ethyl]-5′-O-DMT-5-methyluridine (8)

Compound 5 (0.27 g, 0.95 mmole) was dissolved in anhydrous DMF (3 mL) atroom temperature. To this, compound 4 (0.52 g, 0.86 mmol) and thentriethylamine (0.12 ml, 0.86 mmole) was added, and the reaction wasstirred at room temperature for 4 h. The reaction was quenched byaddition of 5% NaHCO₃ solution (40 mL), extracted with EtOAc (2×60 mL)and the combined organic phases were dried (Na₂SO₄) filtered andevaporated. The crude product was purified by flash columnchromatography with EtOAc as eluant to afford 8 (0.480 g, 66%).

1H NMR (200 MHz, CDCl₃): 11.70 (s, 1H), 8.60 (t, 1H), 8.51 (s, 1H),7.66(s, 1H), 7.39-7.27 (m, 9H), 6.84 (d, 4H), 5.97 (d, 1H), 4.50-3.70(m, 16H), 3.50 (m, 2H), 2.73 (m, 4H), 1.37 (s, 3H); ¹³C-NMR (80 MHz,CDCl₃): 164.50, 162.92, 158.70, 156.06, 153.02, 150.83, 144.43, 135.46,135.29, 130.15, 128.06, 127.72, 127.18, 117.50, 116.88, 113.32, 111.09,87.14, 86.82, 83.50, 82.40, 69.19, 68.94, 62.35, 61.09, 59.89, 55.29,40.88, 18.05, 17.92, 11.87; HRMS (FAB): Calcd for C₄₂H₄₅N₇O₁₂Na⁺862.3024, Found: 862.2991.

Example 9 Synthesis of2′-O-[(N′,N′,-bis-CEOC-guanidinium)ethyl]-5′-O-DMT-5-methyluridine-3′-O-[(2-cyanoethyl)N,N-diisopropyl]Phosphoramidite(9)

Compound 8 (0.39 g, 0.46 mmol) and diisopropylamine tetrazolide (0.08 g,0.46 mmol) were dried by coevaporation with anhydrous MeCN. The residuewas redissolved in anhydrous MeCN (3 mL).2-Cyanoethyl-N,N,N′,N′-tetraisopropylphosphordiamidite (0.27 mL, 0.87mmol) was added and the reaction mixture was stirred at roomtemperature, under argon, for 5 h. The solvent was evaporated and thecrude product purified by flash chromatography with EtOAc/Hexanes aseluant to afford 9 (0.31 g, 64%).

31P NMR (80 MHz, CDCl₃): 150.87 and 150.78; HRMS (FAB): Calcd forC₅₁H₆₃O₁₃N₉PCs- 1172.3259, Found: 1172.3203.

Example 10 Synthesis of2′-O-[2-(N,N′-bis-benzoyl-guanidinium)ethyl]-5′-O-DMT-5-methyluridine(10)

Compound 6 (0.5 g, 1.66 mmol) was dissolved in anhydrous DMF (3 mL).Nucleoside 4 (0.5 g, 0.83 mmol) and triethylamine (0.12 mL, 0.83 mmol)were added. The reaction mixture was stirred at room temperature for 5h. The reaction mixture was added to brine (50 mL) and extracted withethyl acetate (2×50 mL). The organic phase was dried over anhydrousNa₂SO₄ and evaporated to provide a yellow solid. The crude product waspurified by flash column chromatography and eluted with ethylacetate:hexane (1:1) to afford 10 (0.66 g, 93% yield).

1H NMR (200 MHz, CDCl₃): 14.50 (s, 1H), 9.79 (br s, 1H), 9.19 (s, 1H),8.25 (d, 2H, J=6.62 Hz), 8.05 (d, 2H, J=6.94 Hz), 7.69 (s, 1H),7.63-7.25 (m, 15H), 6.80 (d, 4H, J=8.8 Hz), 5.99 (S, 1H), 4.92 (m, 1H),4.28 (m, 1H), 4.18-3.80 (m, 6H), 3.78 (s, 6H), 3.50 (m, 2H), 2.86 (d,1H, J=9.01 Hz), 1.35 (s, 3H); ¹³C NMR (50 MHz, CDCl₃): 178.67, 168.43,164.15, 158.73, 157.13, 150.56, 144.42, 135.49, 135.36, 135.12, 133.65,132.11, 131.87, 130.17, 129.93, 129.51, 129.12, 128.06, 127.14, 113.32,111.07, 87.85, 86.83, 83.19, 82.59, 68.96, 61.70, 55.26, 40.88, 11.89;HRMS (FAB): Calcd for C₄₈H₄₈N₅O₁₀- 854.340, Found: 854.3381.

Example 11 Synthesis of2′-O-[2-(N,N′-bis-benzoyl-guanidinium)ethyl]-5-O-DMT-5-methyluridine-3′-O-[(2-cyanoethyl)N,N-diisopropyl]Phosphoramidite(11)

Compound 11 was prepared from compound 10, according to the proceduredescribed in Example 9. Yield: 1.49 g (70%).

1H NMR (200 MHz, CDCl₃): 14.46 (s, 1H), 9.73 (br s, 1H), 8.64 (br s,1H), 8.25 (d, 2H, J=6.63 Hz), 8.02 (d, 2H, J=6.94 Hz), 7.69 (s, 1H),7.63-7.23 (m, 15H), 6.80 (d, 4H, J=8.74 Hz), 6.06 and 6.01 (2×d, 1H),4.60-3.30 (m, 17H), 2.55 (t, 1H, J=7.6 Hz), 2.35 (t, 1H, J=7.5 Hz),1.40-0.80 (m, 15H); ³¹P NMR (80 MHz, CDCl₃): 150.83 and 150.58; HRMS(FAB): Calcd for C₅₇H₆₄N₇O₁₁PCS- 1186.3456, Found: 1186.3410.

Example 12 Synthesis of2′-O-[2-(N,N′-bis-pivaloylguanidinium)ethyl]-5′-O-DMT-5-methyl Uridine(12)

Compound 12 was prepared from compounds 4 and 7 according to theprocedure described in Example 8. Yield: 0.98 g (73%).

1H NMR (200 MHz, CDCl₃): 13.39 (s, 1H), 9.50 (s, 1H) 9.40 (br s, 1H);7.67 (s, 1H), 7.44-7.20 (m, 9H), 6.83 (d, 4H, J=8.84 Hz), 5.95 (s, 1H),4.50 (m, 1H), 4.20-4.00 (m, 2H), 3.90-3.60 (m, 4H), 3.78 (s, 6H), 3.55(m, 2H), 2.78 (d, 1H, J=8.88 Hz), 1.37 (s, 3H), 1.26 (s, 9H), 1.18 (s,9H); ¹³C NMR (50 MHz, CDCl₃): 193.82, 181.73, 164.12, 158.75, 156.57,150.42, 144.39, 135.49, 135.34, 135.16, 130.17, 128.22, 128.04, 127.15,113.32, 111.03, 87.76, 86.85, 83.17, 82.46, 68.94, 61.73, 60.42, 55.26,42.01, 40.42, 27.90, 27.09, 11.87; HRMS (FAB): Calcd for C₄₄H₅₅N₅O₁₀-814.4027, Found: 814.4054.

Example 13 Synthesis of2′-O-[2-(N,N′-bis-pivaloylguanidinium)ethyl]-5′-O-DMT-5-methyluridine-3′-O-[(2-cyanoethyl)N,N-diisopropyl]Phosphoramidite(13)

Compound 13 was prepared from compound 12, as described in Example 9.Yield: 0.840 g (72%).

1H NMR (200 MHz, CDCl₃): 13.35 (s, 1H), 9.31 (s, 1H), 8.95 (br s, 1H),7.72 and 7.65 (2×s, 1H), 7.45-7.20 (m, 9H), 6.86 (m, 4H), 6.00 and 5.95(2×d, 1H), 4.60-3.20 (m, 17H), 2.63 (t, 1H, J=7.72 Hz), 2.37 (t, 1H,J=7.2 Hz), 1.40-0.80 (m, 33H); ³¹P NMR (80 MHz, CDCl₃): 150.73 and150.55; HRMS (FAB): Calcd for C₅₃H₇₂N₇O₁₁PCs- 1146.4082, Found:1146.4034.

Example 14 Synthesis of5-O-DMT-2′-O-[2-methoxyethyl]-5-(3-aminoprop-1-yne) Uridine (14)

2′-O-Methoxyethyluridine (synthesized according to the proceduresdescribed in U.S. Pat. No. 5,760,202) was treated with an excess ofpyridine/benzoyl chloride to give 3′,5′-dibenzoyl-2′-O-methoxyethyluridine in quantitative yield. This compound was treated with lithiumiodide (1 equivalent) and ceric ammonium nitrate (3 equivalents) inacetonitrile as the solvent. After stirring overnight, TLC indicated theformation of 5-iodo-3′-5′-dibenzoyl-2′-O-methoxyethyl-uridine.Acetonitrile was evaporated and the residue redissolved in CH₂Cl₂ andextracted with saturated NaHCO₃ solution. The organic layer was dried(Na₂SO₄) and evaporated to dryness. The residue was purified on a silicacolumn and eluted with EtOAc/Hexane (7:3). Fractions containing theproduct were pooled together and concentrated.N-Trifluoro-acetylpropargylamine (3 equivalents) was been added, to thiscompound along with tetrakis(triphylphosphine) palladium(0) [(Ph₃P)₄Pd], copper iodide, triethylamine and DMF to provide theC-5-(N-trifluoroacetyl)-propargylamine derivative. Treatment of thiscompound with sodium in methanol afforded the 3′-5′-bis-hydroxyl parentcompound. This compound was 5′-dimethoxy tritylated. The trifluoroacetylgroup was deprotected by treatment with NH₄OH/pyridine.

Example 15 Synthesis of5′-O-DMT-2′-O′[2-(methoxy)ethyl]-5-[3-(N,N′-bis-CEOC-guanidinium)prop-1-yne]Uridine(15)

Compound 15 was prepared from compounds 5 and 14, according to theprocedure described in Example 8. Yield: 0.54 g (17%).

1H NMR (200 MHz, CDCl₃): 11.61 (s, 1H), 9.80 (br s, 1H), 8.13 (d, 2H),7.45-7.20 (m, 9H), 6.83 (d, 4H, J=8.82 Hz), 5.93 (d, 1H, J=3.48 Hz),4.45 (br s, 1H), 4.16-3.96 (m, 6H), 3.77 (s, 6H), 3.82-3.39 (m, 6H),3.37 (s, 3H), 2.73 (q, 4H, J=6.3 Hz); ¹³C NMR (50 MHz, CDCl₃): 162.07,161.77, 158.53, 155.03, 152.67, 149.60, 149.39, 144.64, 143.22, 136.25,135.62, 135.40, 130.03, 128.08, 127.90, 126.88, 123.77, 117.07, 116.20,113.38, 99.27, 88.05,86.96, 83.97, 83.12, 74.84, 71.84, 70.39, 62.35,60.86, 60.44, 60.08, 59.02, 55.32, 31.71, 18.1.

Example 16 Synthesis of5′-O-DMT-2′-O′-[2-(methoxy)ethyl]-5-[3-(N,N′-bis-CEOC-guanidinium)prop-1-yne]uridine-3′-O-[(2-cyanoethyl)N,N-diisopropyl]phosphoramidite(16)

Compound 16 was prepared from compound 15, according to the proceduredescribed in Example 9. Yield: 0.24 g (35%).

31P NMR (80 MHz, CDCl₃): 150.91 and 150.15; HRMS (FAB): Calcd forC₅₄H₆₄O₁₄N₉PNa- 1116.4208, Found: 1116.4243.

Example 17 Synthesis of5′-O-DMT-2′-O′[2-(methoxy)ethyl]-5-[3-(N,N′-bis-benzoylguanidinium)prop-1-yne]uridine(17)

Compound 17 was prepared from compounds 6 and 14, according to theprocedure described in Example 8. Yield: 0.86 g (38%).

1H NMR (200 MHz, CDCl₃): 14.34 (s, 1H), 9.57 (br s, 1H), 9.29 (t, 1H,J=4.88 Hz), 8.24 (d, 2H, J=6.62 Hz), 8.20 (s, 1H), 8.02 (d, 2H, J=6.72Hz), 7.63-7.19 (m, 15H), 6.82 (d, 4H, J=8.84 Hz), 5.95 (d, 1H, J=3.3Hz), 4.47 (br s, 1H), 4.26-4.00 (m, 5H), 3.84-3.37 (m, 14H), 2.10 (br s,1H); ¹³C NMR (50 M Hz, CDCl₃): 178.62, 167.83, 161.65, 158.60, 156.06,149.43, 144.72, 140.75, 137.41, 135.60, 135.37, 133.55, 132.12, 131.93,130.05, 129.65, 129.10, 128.00, 126.88, 113.35, 99.66, 88.71, 88.11,86.95, 83.93, 83.08, 74.54, 71.37, 70.32, 69.06, 62.28, 58.96, 55.19,31.62; HRMS (FAB): Calcd for C₅₁H₄₉O₁₁N₅- 908.3507, Found: 908.3546.

Example 18 Synthesis of5′-O-DMT-2′-O-[2-(methoxy)ethyl]-5-[3-(N,N′-bis-benzoylguanidinium)prop-1-yne]uridine-3′-O-[(2-cyanoethyl)N,N-diisopropyl]phosphoramidite(18)

Compound 18 was prepared from compound 17, according to the proceduredescribed in Example 9. Yield: 0.72 g (86%).

31PNMR (80 MHz, CDCl₃): 150.93 and 150.21; HRMS (FAB): Calcd forC₆₀H₆₆N₇O₁₂P- 1108.4585, Found: 1108.4548.

Example 19 Synthesis of 5-(3-phthalimidopropy-1-nyl)-2′-deoxyuridine(19)

To a suspension of 5-iodo-2′-deoxyuridine (2 mmol) in 10 mL of CH₂Cl₁,is added trifluoroacetic anhydride (5 mmol) at room temperature. Themixture is stirred overnight. After concentration of the mixture, themixture is dried in vacuo at room temperature to give a solid foam of3′,5′-di-O-trifluoroacetyl-5-iodo-2′-deoxyuridine. To a mixture of3′,5′-di-O-trifluoroacetyl-5-iodo-2′-deoxyuridine (5 mmol) andN-1-phthalimidoprop-1-yne (10 mmol), tetrakis (triphenylphosphine)palladium(0) (0.2 mmol), copper(I) iodide (0.3 mmol) and triethylamine(6 mmol) are added in 10 ml DMF. The mixture is stirred at roomtemperature for 18 hours and then concentrated in vacuo. To theconcentrate, AG-1×8 anion exchange resin (HCO₃ ⁻ form, 3 equivalents),20 mL of methanol and 20 mL of CH₂Cl₂ are added and the suspension isstirred for 1 h. The residue is purified by silica column chromatographyto afford 5-(3-phthalimidoprop-1-ynyl)-2′-deoxyuridine (19).

Example 20 Synthesis of5′-O-DMT-5-(3-phthalimidoprop-1-nyl)-2′-deoxy-uridine (20)

5-(3-Phthalimidoprop-1-nyl)-2′-deoxyuridine (19) is treated with 1.2equivalents of 4,4′-dimethoxytrityl chloride in pyridine containing 0.1equivalent of dimethylaminopyridine. After stirring for 4 h, pyridine isevaporated and the residue dissolved in methylene chloride, washed withsaturated sodium bicarbonate solution, dried over anhydrous sodiumsulfate and concentrated. The residue is purified using silica columnchromatography using ethyl acetate:hexanes to yield the 5′-O-DMTderivative (20).

Example 21 Synthesis of 5′-O-DMT-2′-deoxy-5-[3-(amino)prop-1-yne]uridine(21)

The above phthalimido compound (20), on hydrazine treatment, yieldedamino compound 21 according to the procedure described in Example 4.

Example 22 Synthesis of5′-O-DMT-2′-deoxy-5-[3-(N,N′-bis-CEOCguanidinium)-prop-1-yne]uridine(22)

Compound 22 was prepared from compound 21 according to the proceduredescribed in Example 8. Yield: 3.47 g (63%).

1H NMR (200 MHz, CDCl₃): 11.59 (s, 1H), 9.80 (br s, 1H), 8.22 (br s,1H), 8.11 (s, 1H), 7.43-7.20 (m, 9H), 6.82 (d, 4H, J=8.68 Hz), 6.28 (t,1H, J=6.15 Hz), 4.50 (br s, 1H), 4.29 (m, 4H), 4.11 (m, 3H), 3.75 (s,6H), 3.40 (m, 3H), 2.69 (m, 4H), 2.50 (m, 1H), 2.30 (m, 1H); ¹³C NMR (50MHz, CDCl₃): 162.92, 162.24, 158.82, 155.41, 152.88, 149.60, 144.89,143.87, 135.79, 135.68, 130.28, 128.26, 127.20, 117.43, 116.64, 113.54,99.40, 88.27, 87.20, 86.88, 86.08, 75.17, 72.28, 63.83, 61.09, 60.29,55.54, 41.75, 31.99, 18.30, 18.11; HRMS (FAB): Calcd for C₄₂H₄₂N₇O₁₁-820.2942, Found: 820.2918.

Example 23 Synthesis of5′-O-DMT-2′-deoxy-5-[3-(N,N′-bis-CEOCguanidinium)-prop-1-yne]uridine-3′-O-[(2-cyanoethyl)N,N-diisopropyl]phosphoramidite(23)

Compound 23 was prepared from compound 22 according to the proceduredescribed in Example 9. Yield: 1.43 g (58%).

31P NMR (80 MHz, CDCl₃): 149.56 and 149.26; HRMS (FAB): Calcd forC₅₁H₅₈N₉O₁₂PNa- 1042.3840, Found: 1042.3808.

Example 24 Synthesis of5′-O-DMT-2′-O-[2-(N-,N′-CEOC-guanidinium)ethyl]-3-O-succinyl-5-methyluridine(24)

Compound 8 (0.252 g, 0.30 mmol) was co-evaporated with anhydrousacetonitrile. To this, succinic anhydride (0.6 g, 0.6 mmol), DMAP (0.018g, 0.15 mmol), anhydrous pyridine (0.048 mL, 0.6 mmol) and CH₂Cl₂ (1 mL)were added and stirred at room temperature under an inert atmosphere for4 h. The reaction mixture was diluted with CH₂Cl₂ (25 mL) and washedwith cold 10% aqueous citric acid (20 mL) and brine (25 mL). The organicphase was dried over anhydrous Na₂SO₄ and concentrated to providecompound 22 (0.248 g, 88%) as a foam. R_(f) (0.25, 5% MeOH in CH₂Cl₂).

1H NMR (200 MHz, CDCl₃): 1.34 (s, 3H), 2.2-2.5 (m, 8H), 3.45 (d, 1H,J=5.4 Hz), 3.64 (d, 1H, J=11.62 Hz), 3.72-4 (m, 10H), 4.15 (br s, 1H),4.26-4.38 (m, 6H), 5.40 (d, 1H, J=4.4 Hz), 5.93 (d, 1H, J=2.12 Hz), 6.83(d, 4H, J=7.22 Hz), 7.24-7.4 (m, 9H), 7.77 (s, 1H), 8.57 (s, 1H), 10.51(s, 1H); HRMS (FAB): Calcd for C₄₆H₅₀N₇O₁₅ 940.3365, Found: 940.3346.

Example 25 Synthesis of5′-O-DMT-2′-O-[2-(N,N′-CEOC-guanidinium)ethyl]-5-methyluridine-3′-O-succinyl-CPG(25)

Compound 24 (0.227 g, 0.24 mmol) was dried over P₂O₅ in vacuo at 40° C.overnight. Anhydrous DMF (0.62 mL) was added followed by2-(1H-benzotriazole)-1-yl)-1,1,3,3-tetramethyluroniumtetrafluoroborate(0.077 g, 0.24 mmol) and N-methylmorpholine (53 mL, 0.48 mmol). Thereaction mixture was vortexed to obtain a clear solution. To thissolution, anhydrous DMF (2.38 mL) and activated CPG (1.03 g, 115.2mmol/g, particle size 120/200, mean pore diameter 520 Å) were added. Thereaction mixture was then allowed to shake on a shaker for 18 h. Analiquot was withdrawn and the loading capacity was estimated.Functionalized CPG was filtered and washed thoroughly with DMF, CH₃CNand Et₂O. It was then dried in vacuo overnight. Functionalized CPG (23)was then suspended in a capping solution (aceticanhydride/lutidine/N-methylimidazole in THF, PerSeptive Biosystems,Inc.) and allowed to shake on a shaker for 2 h. Functionalized CPG wasthen filtered, washed with CH₃CN and Et₂O, dried in vacuo and theloading capacity determined by standard procedure. Final loading was32.8 mol/g.

Example 26 Synthesis of oligonucleotides containing2′-O-[2-(guanidinium)-ethyl] Modification and Deprotection of CEOC Groupusing a Novel Procedure

The amidite 9 was dissolved in anhydrous acetonitrile to obtain a 0.1 Msolution and loaded onto a Expedite Nucleic Acid Synthesis system(Millipore 8909) to synthesize the oligonucleotides. The couplingefficiencies were more than 98%. For the coupling of the modifiedamidite (9) coupling time was extended to 10 minutes and this step wascarried out twice. All other steps in the protocol supplied by Milliporewere used as such. After completion of the synthesis, CPG was suspendedin 50% piperidine in water and kept at room temperature for 24 h todeprotect the CEOC protecting group on the guanidino group. Under thesame conditions, oligonucleotides are cleaved from CPG. The solvent wasthen evaporated and the CPG treated with aqueous ammonia solution (30 wt%) at 55° C. for 6 h to complete the deprotection of exocyclic aminoprotecting groups. This was purified on High Performance LiquidChromatography (HPLC, Waters, C-4, 7.8×300 mm, A=50 mM triethylammoniumacetate, pH=7, B=acetonitrile, 5 to 60% B in 55 Min, Flow 2.5mL/min.=260 nm). Detritylation with aqueous 80% acetic acid andevaporation, followed by desalting by HPLC on Waters C-4 column, gave2′-modified oligonucleotides (Table I). Oligonucleotides were analyzedby HPLC, CGE and mass spectrometry.

TABLE I Oligonucleotides containing 2′-O-[2-(guanidinium) ethylmodification HPLC Mass Mass Ret. Calcu- Ob- Time SEQ ID No. Sequence(5′-3′) lated served (min^(a))  1 T*CC AGG T*GT* 5238.22 5238.21 18.69CCG CAT* C  2 CTC GTA CT*T* 5797.49 5797.15 18.06 T*T*C CGG TCC  3 TTTTTT TTT TTT 6122.93 6122.60 18.21 TTT T*T*T* T* 13 TTT TTT TTT TTT5920.59 5920.21 19.32 TTT TT*T T*  7 TTT TTT TTT TTT 5818.47 5818.6219.46 TTT TTT T*  8 T*T*T* T*T*C TCT 4931.41 4931.46 21.93 CTC TCT  9TT*T TT*C TCT* CTC 4830.8 4830.29 16.07 T*CT 10 T*TT* TT*C TCT CTC4729.73 4729.47 19.11 TCT Ret. time = retention time (in minutes); T* =2′-O-[2-(guanidinium)ethyl] ^(5Me)U ^(a)Waters C-4, 3.9 × 300 mm,solvent A = 50 mm TEAAc, pH 7; Solvent B = CH₃CN; gradient 5-60% B in 55min; flow rate 1.5 mL/min., l = 260 nm

TABLE II Tm values of 2′-O-[2-(2-guanidiniumethyl)oxyethyl] modifications Target Sequence RNA ΔTm ΔTm/ SEQ ID No. 5′-3′° C. ° C. mod. 11 TCC AGG TGT CCG 62.3 — CAT C  1 T*CC AGG T*GT* 70.58.2 2.05 CCG CAT* C 12 CTC GTA CTT TTC 61.8 CGG TCC  2 CTC GTA CT*T*61.28 −0.53 −0.13 T*T*C CGG TCC T* = 2′-O-[2-(guanidinium)ethyl ^(5Me)U.The ΔTm/modification relative to 2′-deoxy P = S is obtained by adding0.8 to the value shown in last column. This value relative to othermodifications is shown in FIGS. 2 and 3.

2′-O-Guanidiniumethyl modification shows the highest binding affinitychange (ΔTm/mod.) among 2′-modifications studied so far. This point isillustrated in FIG. 2.

As shown in FIG. 3, the ΔTm/modification depends on the nature ofplacement of the cationic modification. There could be charge repulsionwhen the cationic groups are placed close to each other.

Example 27 Synthesis ofN′-Benzoyl-2′-deoxy-5′-O-(4,4′-dimethoxytrityl)-cytidine (26)

2′-Deoxycytidine (5.82 g, 25.6 mmol) was dried by evaporation and takenup in dry pyridine (100 mL). To this suspension, trimethylsilyl chloride(11.5 mL, 90.6 mmol) was added and the mixture was stirred at ambienttemperature for 2 h. The reaction mixture was cooled in an ice-waterbath and benzoyl chloride (4.60 mL, 39.6 mmol) was slowly introduced.The ice-waterbath was removed and the mixture was stirred at roomtemperature for additional 2 h. The reaction was quenched by addingmethanol (15 mL), concentrated to one half of the original volume andfiltered. To the filtrate, water (30 mL) was added and the solution wasevaporated to an oil. Further evaporation with water (3×30 mL) wasperformed to remove pyridine, and the resulting residue was partitionedbetween water and ethyl acetate. After vigorous stirring, the productcrystallized from the aqueous layer. The product crystals were washedwith cold water and ethyl acetate and were used in the next step withoutfurther purification. Crude N⁴-benzoyl-2′-deoxycytidine was dried byevaporation with dry pyridine (3×50 mL) and then dissolved in the samesolvent (100 mL). 4,4-Dimethoxytritylchloride (7.4 g, 22 mmol) was addedportion-wise to the reaction mixture and the stirring was continuedovernight at ambient temperature. The reaction mixture was evaporated toafford an oil and dissolved in dichloromethane (100 mL). The organicphase was washed with saturated aqueous NaHCO₃ (50 mL) and water (3×100mL) and dried with anhydrous Na₂SO₄. After coevaporation with toluene,the residue was applied onto a silica gel column and eluted with agradient of MeOH in CH₂Cl₂ (0-8% MeOH). The product (25) was obtained in65% yield (9.0 g) starting from 2′-deoxycytidine.

1H NMR (200 MHz, CDCl₃): 8.30 (s, 1H, d, J=7.6), 7.45-7.10 (m, 9H), 6.84(m, 4H), 7.90-7.40 (m, 5H), 6.31 (t, 1H, J=5.9, Hz), 4.55 (m, 1H), 4.19(m, 1H), 3.78 (s, 6H), 3.49 (dd, 1H, J=3.2 and 11.0 Hz), 3.42 (dd, 1H,J=3.9 and 10.8 Hz), 2.77 (m, 1H).

Example 28 Synthesis of2′-deoxy-N′-[2-(amino)ethyl]-5′-O-(4,4′-dimethoxy-trityl)cytidine (27)

A solution of ethylenediamine (96.9 mmol) in 2-propanol (10 mL) is addedto compound 25 (2.61 g, 4.12 mmol) and stirred until the mixture isclear. 1,5,7-Triazabicyclo[4.4.0]dec-5-ene (TBD, 2.40 g, 17.2 mmol) isadded and the reaction mixture is stirred at ambient temperature for 48h. The reaction mixture is evaporated to afford an oil, dissolved inCHCl₃ (100 mL), extracted with 0.1 mol L⁻¹ aq. NaOH (2×50 mL) and water(4×50 mL). The organic phase is dried with anhydrous Na₂SO₄, evaporatedand dissolved in CH₂Cl₂. Silica gel column purification yields the pureproduct 27.

Example 29 Synthesis of2′-deoxy-5′-O-(4,4′-dimethoxytrityl-N′-[2-(N,N′-bis-CEOC-guanidinium)ethyl]cytidine(28)

Compound 27, on treatment with reagent 5, gives compound 28 as describedin Example 8.

Example 30 Synthesis of2′-deoxy-5′-O-(4,4′-dimethoxytrityl)-N′-[2-(N,N′-bis-CEOC-guanidinium)ethyl]cytidine-3′-O-[(2-cyanoethyl)N,N-diisopropyl)Phosphoramidite (29)

Compound 29 (0.29 mmol) is dried by evaporation with dry acetonitrile(3-20 mL) and finally in vacuo for 30 minutes.2-Cyanoethyl-N,N,N′,N′-tetraisopropylphosphorodiamidite (0.12 mL, 0.38mmol) and dry acetonitrile (1.0 mL) are added and the mixture is stirreduntil all material dissolves. 1H-Tetrazole (0.45 mol L⁻¹ in MeCN, 0.64mL, 0.29 mmol) is added and the reaction mixture is shaken. After onehour at ambient temperature, 100 mL of saturated aqueous NaHCO₃ solutionis added to the reaction mixture and the resulting solution is extractedwith ethyl acetate (2×40 mL). The organic phase is dried with anhydrousNa₂SO₄ and evaporated to dryness to afford pure compound 29.

Example 31 Synthesis of5′-O-(4,4′-dimethoxytrityl)-5-(3-aminoprop-1-nyl)-N4-benzoyl-2′-deoxycytidine(33)

To 5-iodo-2′-deoxycytidine (5 mmol), tetrakis (triphenylphosphine)palladium(0) (0.5 mmol), copper(I) iodide (1 mmol) andN-1-phthalimidoprop-3-yne (12 mmol) in 15 mL of dry DMF, 10 mmol oftriethylamine is added. After stirring for 18 h, AG-1×8 anion exchangeresin (HCO₃ ⁻ form, 3 equivalents), 20 mL of methanol, and 20 mL ofCH₂Cl₂ are added, and the suspension is stirred for 1 h. The reaction isfiltered through a sintered glass funnel, and the DMF removed in vacuo.Flash chromatography yielded the product (30). This compound is treatedwith benzoic anhydride (one equivalent) in dry pyridine to giveN⁴-benzoyl derivative (31). To a mixture of N⁴-benzoyl derivative and4,4′-dimethoxytritylchloride, dry pyridine is added to afford the5′-dimethoxytrityl derivative (32), which is then converted into aminocompound 33 according to the procedure described in Example 4.

Example 32 Synthesis of5′-O-(4,4′-dimethoxytrityl)-2-[2-(N,N′-bis-CEOCguanidinium)ethyl]amino-2′-deoxyadenosine-3′-O-[(2-cyanoethyl)N,N-diisopropyl]phosphoramidite(34)

2-Fluoroadenosine is synthesized according to the procedure described byKrolikiewicz and Vorbruggen (Nucleosides & Nucleotides 13, 673, 1994).Ethylenediamine (5 equivalents in 2-methoxyethanol) is added to2-fluoroadenosine and heated at 100°. The resulting 2-(aminoethyl)derivative is then converted into 2-(2-guanidinium)ethyl derivative withreagent 5 as described in example 8. This was then treated with benzoylchloride under transient protection conditions and thendimethoxytritylated at the 5′-position. The resulting compound was thenphosphitylated following the procedures described above to give theadenosine derivative 36 functionalized at the 2-position.

Example 33 Synthesis of5′-O-(4,4′-dimethoxytrityl)-2-[2-(bis-N,N-CEOCguanidinium)ethyl]-2′-deoxyguanosine-3′-O-[(2-cyanoethyl)N,N-diisopropyl]phosphoramidite (34)

2-Fluoroinosine is synthesized according to the procedure described byKrolikiewicz and Vorbruggen (Nucleosides & Nucleotides 13, 673, 1994).Ethylenediamine (5 equivalents in 2-methoxyethanol) is added to2-fluoroinosine and heated at 100° C. The free amine is guanylated onthe side chain at the 2-position by treatment with reagent 5 asdescribed in Example 8. The product is first dimethoxytritylated at the5′-position, then phosphitylated following the procedures describedabove to give the guanosine derivative functionalized at the 2-position(37).

Example 34 Synthesis of5′-O-(4,4′-dimethoxytrityl)-N²-isobutyryl-N⁶-[2-(bis-N,N′-CEOC-guanidinium)ethyl)-2-aminoadenosine-3′-O-[(2-cyanoethyl)N,N-diisopropyl]phosphoramidite(35)

To 2′-deoxyguanosine (2 mmol), dried by coevaporation with pyridine,suspended in 40 mL of pyridine and cooled in an ice bath under an argonatmosphere, trifluoroacetic anhydride (16 mmol) is added following theprocedure of Kung and Jones (Tetraheron Lett., 32, 3919, 1991). After 40minutes, ethylenediamine (30 mmol) is added and stirred for 24 h at roomtemperature. The reaction mixture is concentrated and purified by silicacolumn chromatography. The product is guanylated with reagent 5 as inExample 8 and protected at N²-position with an isobutyryl group bytreating with isobutyric anhydride, 5′-dimethoxytritylated at the5′-position and purified. The resulting compound is treated with benzoicanhydride (N⁶ protection) and then phosphitylated following theprocedures described above to afford the 2,6-diaminopurine derivativefunctionalized at the 6-position (35).

Example 35 Nuclease Stability Assay of 2′-O-[2-(guanidinium)ethyl]oligonucleotides (SEQ ID No. 3: ISIS 109990, ISIS 109989 and ISIS109973)

The stability of the oligomers was tested by incubating them with snakevenom phosphodiesterase (Phosphodiesterase I, USB # 20240), anon-sequence specific 3′-exonuclease. Typical Nuclease digestion:

-   -   70 uL nanopure water    -   10 uL 1 uM ³²P labeled oligonucleotide    -   10 uL 10× buffer    -   10 uL SVPD @ 5×10⁻² Units/mL    -   100 uL        Final reaction conditions:

-   Buffer    -   50 mM Tris.HCl, pH 8.0    -   75 mM NaCl    -   14 mM MgCl₂

-   Oligomer concentration    -   100 nM

-   Enzyme concentration    -   5×10⁻³ Units/mL

The reactions were incubated at 37° C. Aliquots (5 μL) were removed overa set time course and transferred to tubes containing 2× TBE informamide with a trace of Bromphenol Blue, then frozen at B4° C. untilanalysis. The reactions were analyzed by PAGE/Phosphorimaging. Samplesat specific time points were centrifuged briefly to settle the samples,then loaded on a 20% acrylamide gel. After running, the gels wereexposed and read with a Molecular Dynamics Phosphorimager (FIG. 4). The% full length (N) of oligomer in each lane was calculated.

As shown in FIG. 4, the placement of guanidinium residue protects theoligonucleotide phosphodiester internucleotide linkage againstexonuclease degradation. With increasing number of substituents, thenuclease resistance also increases. Placement of four guanidiumsubstituents in a 19mer at the 3′-end fully protects the oligomer.

Example 36 Synthesis of 2′-O-(2-guanidinium)-ethyl Oligonucleotides forTriplex Formation

The sequence specific recognition of duplex DNA by pyrimidineoligonucleotides involves the formation of triple helical structureswhich are stabilized by Hoogsteen hydrogen bonds between the bases on aDNA target and pyrimidine third strand. (Neidel, Anti-Cancer Drug Des.12, 433-442, 1997; Giovannangeli and Helene, Antisense Nulceic Acid DrugDev. 7, 413-421, 1997; Maher, Cancer Invest. 14, 66-82, 1996.)

Examination of a molecular model of triple helix with a RNA third strandindicates that the 2′-hydroxyl groups of RNA and the phosphate groups ofthe DNA second strand are in close proximity. (Hélène, et al., NucleicAcid Res. 21, 5547-5553, 1993.) 2′-Aminoethoxy modified oligonucleotidesstabilize the triplex formation. (Cuenourd et al., Angew. Chem. Int.Ed., 37, 1288-1291, 1998.) 2′-Guanidinoethyl modified oligonucleotides,by virtue of its stabilized multi-point charge site, would stabilizetriplex formation. Following duplex DNA was targeted with2′-guanidiniumethyl-modified oligonucleotides (SEQ ID No. 04: ISIS113254, ISIS 113929 and ISIS 113255) used as strand 3 for triplexformation.

Strand 2 5′-GCT AAA AAG AGA GAG AGA TCG-3′ SEQ ID No. 05 Strand 1 5′-CGATTT TTC TCT CTC TCT AGC-5′ SEQ ID No. 06

Example 37 Synthesis of Compounds 40 and 41

Compound 37 is synthesized according to the procedure described byBehrens et al. (Bioorg. Med. Chem. Lett., 1995, 5, 1785). Compound 37 isconverted to the guanidinium derivative 38 using reagent 5 as describedin Example 8. Compound 38 is then treated with 1 equivalent of4,4′-dimethoxytrityl chloride and pyridine to form compound 39, which isthen phosphitylated to yield compound 40. Synthesis of an oligomer usingcompound 40 results in an oligomer having the guanidinium group at the5′ end.

Also, compound 39 is succinylated and coupled to controlled pore glass(CPG) to yield compound 41. Synthesis of an oligomer using compound 41results in an oligomer having the guanidinium group at the 3′ end.

Example 38 Synthesis of Compounds 49 and 56

(FIGS. 19-23)

Compound 42 is synthesized according to the reported procedures (seeMigawa et al., Synthetic Communications, 1996, 26, 3317-3322).Glycosylation of 42 with chloro sugar 43 according to a previouslyreported procedure (Ramasamy et al., Chem. Soc. Perkin Trans., I, 1989,2375-2384) gives nucleoside 44 which is then acylated at the N² positionto give the completely protected nucleoside 45. Compound 45 is thenreduced to the 7-aminomethyl derivative 46 in the presence of palladiumand hydrogen gas at 50 psi. Guanylation of 46 with guanylating reagent 5in DMF and triethyl amine gives 48 which is then subject todesilylation, selective tritylation at the 5′ position andphosphitylation at the 3′ position to give compound 49.

Compound 50 (prepared as per the procedure of: Porcari et al.,Nucleosides and Nucleotides, 1999, 18, 153-159) is silylated withMarkovick reagent in pyridine to give Compound 51. Compound 51 is thenacylated at the 4-position of the exocyclic amino group with benzylchloride and pyridine to give the bisbenzoyl derivative which isconverted to mono benzoyl derivitive, compound 52, by treatment withaqueous ammonia. Deoxygenation of 52 at the 2′ position gives compound53. Catalytic hydrogenation of 53 in the presence of Palladium andhydrogen gives compound 54 which is converted into the guanidinoderivative 55 according to the procedure used for 5. Compound 55 ondesilylation, tritylation at 5′-position and phosphitylation at3′-position gives compound 56.

Example 39

TABLE III Triplex binding affinity enhancement of 2′-O-GE modificationagainst double stranded DNA II III IV T* TM ° C. ΔTm/mod/° C. TM ° C.ΔTm/mod/° C. TM ° C. ΔTm/mod/° C. 2′-deoxy 21.9 2′-O-AE 35.9 3.52′-O-AP* 32.4 2.1 2′-O-GE 29.3 2.53 32.4 2.65 43.6 4.12

Oligomers II, III and IV each have SEQ ID NO. 4 (TTT TTC TCT CTC TCT)with various groups attached at various 2′-positions ((2′-H,2′-O-aminoethyl (2′-O-AE); 2′-aminopropyl (2′-O-AP); and2′-guanidiniumethyl (2′-O-GE)). Oligomer II is modified at the 1,3 and 5positions (SEQ ID NO. 14-17 T*TT* TT*C TCT CTC TCT); oligomer III ismodified at the 2, 5, 9 and 12 positions (SEQ ID NO. 18-21 TT*T TT*CTCT* CTC T*CT); and oligomer IV is modified at the 1-5 positions (SEQ IDNO. 22-25 T*T*T* T*T*C TCT CTC TCT).

The results of the study show that the 2′-O-GE modification has impartshigher binding affinity to the resulting modified oligomers.

1. A monomer of the formula:

wherein: Y₁ is a hydroxyl protecting group; Y₂ is a phosphoramidite,H-phosphonate, phosphate triester, or a linking moiety attached to asolid support; each R₂, R₃, R_(3′), and R₄ is, independently, hydrogen,C(O)R₅, substituted or unsubstituted C₁-C₁₀ alkyl, substituted orunsubstituted C₂-C₁₀ alkenyl, substituted or unsubstituted C₂-C₁₀alkynyl, alkylsulfonyl, or arylsulfonyl, wherein the substituent groupsare selected from hydroxyl, amino, alkoxy, carboxy, benzyl, phenyl,nitro, thiol, thioalkoxy, halogen, alkyl, aryl, alkenyl and alkynyl; orR₃ and R₄, together, are R₇; each R₅ is, independently, substituted orunsubstituted C₁-C₁₀ alkyl, trifluoromethyl, cyanoethyloxy, methoxy,ethoxy, t-butoxy, allyloxy, 9-fluorenylmethoxy,2-(trimethylsilyl)-ethoxy, 2,2,2-trichloroethoxy, benzyloxy, butyryl;iso-butyryl, phenyl or aryl; R₇ is hydrogen or forms a phthalimidemoiety with the nitrogen atom to which it is attached; and n is aninteger from 1 to about
 6. 2. The compound of claim 1 wherein Y₁ isdimethoxytrityl.
 3. The compound of claim 1 wherein Y₁ ismonomethoxytrityl.
 4. The compound of claim 1 wherein Y₂ is a linkingmoiety attached to a solid support.
 5. The compound of claim 1 whereinY₂ is succinyl controlled pore glass (CPG).